Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota 55108, United States.
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States.
J Agric Food Chem. 2023 Jun 21;71(24):9481-9489. doi: 10.1021/acs.jafc.3c01220. Epub 2023 Jun 6.
Thermal processing (e.g., pasteurization and sterilization) is a critical step ensuring the microbial safety of our foods. Previous work from our laboratory has examined the covalent reactions occurring between proteins and a broad selection of flavor compounds under ambient storage temperatures (25-45 °C). However, similar research on reactions of flavor compounds with a protein under thermal processing conditions has not been investigated. In the current study, covalent adduct formation between β-lactoglobulin (BLG) and 46 flavor compounds encompassing 13 different classes of functional groups was investigated under pasteurization and sterilization conditions by UPLC-ESI-QTOF-MS. BLG was chosen as a representative protein for this study because it is structurally well characterized, its molecular weight is well suited for ESI-MS analysis (18.2 kDa), and it is broadly used in the food industry. Schiff base, aza-Michael addition, and disulfide linkages were the main types of covalent interactions occurring across the reactive samples. Among them, isothiocyanates, aldehydes, and thiol-containing compounds were generally very reactive. Increasing the severity of the thermal treatment [high-temperature-short-time (HTST) pasteurization, in-container pasteurization (IC), and ultra-high-temperature (UHT) sterilization conditions] accelerated the reactions of BLG with flavor compounds, which revealed reactivity of three flavor compounds not previously observed to react at room temperature (eugenol, 4-vinyl phenol, and 3-nonen-2-one). Ketones [other than 2-hydroxy-3-methyl-2-cyclopenten-1-one (cyclotene), diketones, and unsaturated ketones], alcohols, acids, alkenes (terpenes), esters, lactones, 3-acetylpyridine, methyl anthranilate, vanillin, 2-methylthiophene, and dimethyl sulfone did not show measurable reactivity with BLG under the thermal processing conditions examined. An overall view of the data shows that the HTST heat treatment (72 °C for 15 s) had the least effect on the extent of reaction while in-container pasteurization conditions (63 °C for 30 min) produced a similar extent of reaction as the UHT (130 °C 30 s) heat treatment. These varying extents of adductation are in reasonable accord with what one might expect, given that the rates of most classes of chemical reactions occurring near ambient temperature increase by a factor of 2-4 for each increase of 10 K in temperature. Unfortunately, our methodology did not permit us to obtain meaningful data using the most aggressive standard sterilization thermal conditions (110 °C for 30 min) because extensive aggregation/coagulation removed essentially all of the BLG protein from the reaction mixtures prior to MS analysis.
热处理（例如巴氏杀菌和灭菌）是确保食品微生物安全的关键步骤。我们实验室之前的工作研究了在环境储存温度（25-45°C）下，蛋白质与广泛选择的风味化合物之间发生的共价反应。然而，对于在热加工条件下风味化合物与蛋白质的反应，尚未进行类似的研究。在当前的研究中，通过 UPLC-ESI-QTOF-MS 研究了β-乳球蛋白（BLG）与 46 种风味化合物在巴氏杀菌和灭菌条件下的共价加合物形成，这些风味化合物涵盖了 13 种不同类别的官能团。选择 BLG 作为本研究的代表性蛋白质，是因为它的结构特征良好，分子量非常适合 ESI-MS 分析（18.2 kDa），并且在食品工业中广泛使用。席夫碱、氮杂迈克尔加成和二硫键是反应样品中发生的主要共价相互作用类型。其中，异硫氰酸酯、醛和含巯基的化合物通常反应非常活跃。增加热处理的严重程度[高温短时间（HTST）巴氏杀菌、容器内巴氏杀菌（IC）和超高温（UHT）灭菌条件]会加速 BLG 与风味化合物的反应，这揭示了三种风味化合物在室温下未观察到反应的反应性（丁香酚、4-乙烯基苯酚和 3-壬烯-2-酮）。在研究的热加工条件下，酮[除 2-羟基-3-甲基-2-环戊烯-1-酮（环丁烯）、二酮和不饱和酮]、醇、酸、烯烃（萜烯）、酯、内酯、3-乙酰基吡啶、甲基邻氨基苯甲酸酯、香草醛、2-甲基噻吩和二甲亚砜与 BLG 没有表现出可测量的反应性。数据的总体情况表明，HTST 热处理（15 秒 72°C）对反应程度的影响最小，而容器内巴氏杀菌条件（30 分钟 63°C）产生的反应程度与 UHT（30 秒 130°C）相同。这些不同程度的加合物形成与人们的预期基本一致，因为在环境温度附近发生的大多数类化学反应的速率每增加 10 K，就会增加 2-4 倍。不幸的是，我们的方法学不允许我们使用最具侵略性的标准灭菌热条件（30 分钟 110°C）获得有意义的数据，因为广泛的聚集/凝聚在 MS 分析之前基本上将 BLG 蛋白从反应混合物中除去。
