Food Research International 172 (2023) 113162 Available online 19 June 2023 0963-9969/© 2023 Published by Elsevier Ltd. Metabolomics investigation of global responses of Cronobacter sakazakii against common sanitizing in infant formula processing environments Zejia Lin a, * , Guoshu Wang a , Kexin Zhang a , Shaoqian Jiang a , Songshen Li a , Hongshun Yang b a Department of Food Science & Technology, National University of Singapore, Singapore 117542, Singapore b Shaoxing Key Laboratory of Traditional Fermentation Food and Human Health, Jiangnan University (Shaoxing) Industrial Technology Research Institute, Zhejiang 312000, China A R T I C L E INFO Keywords: Cronobacter sakazakii Food safety Dairy products UPLC-MS NMR Sanitization ABSTRACT Cronobacter sakazakii, an opportunistic bacterium, has raised a serious outbreak in powdered infant formula recent years. In this work, four sanitizing strategies used during infant formula processing, including chlorine, quaternary ammonium chloride (QAC), 60 ◦ C heating, and malic acid (MA), were utilized against C. sakazakii among planktonic, air-dried (A), and air-dried & washed (AW) state, followed by an exploration of the metabolic responses induced by these treatments via a dual-platform metabolomics analysis with the ultra-high perfor- mance liquid chromatography-mass spectrometry and nuclear magnetic resonance. In the planktonic state, MA was the most effective in inhibiting bacterial growth, followed by chlorine, QAC, and 60 ◦ C heating. Under A state, the effcacy of heating improved considerably, compared to that in the planktonic state, and remained unaltered under AW state. Chlorine and QAC were ineffective to control bacterial growth under A state, but their effcacy rose under AW state. Furthermore, the metabolomic analysis revealed chlorine induces amino acids catabolism, membrane lysis, and depression in carbohydrate and nucleotide metabolism in both planktonic and AW states, while the initiation of antioxidation mechanism was only found under AW state. Although the metabolic change caused by QAC in the planktonic state was similar to chlorine, the accumulation of osmo- protectant and membrane phospholipids within the AW cells refected the effort to restore intracellular ho- meostasis upon QAC. Heating was characterized by considerable amino acid anabolism, along with mildly perturbed carbohydrate and nucleotide metabolism for heat shock protein preparation in both states. Lastly, MA promoted amino acid-dependent acid resistance under the planktonic state, and the regulation of antioxidation and osmoprotection under AW state. The metabolomics study elucidated the intracellular perturbation induced by common sanitizing, as well as the bacterial response, which provides insights for novel sanitization development. 1. Introduction Cronobacter sakazakii, an foodborne pathogen primarily related with the contamination of powdered infant formula, could cause life- threatening illnesses, including bacteremia, necrotizing enterocolitis, and neonatal meningitis in neonates as well as preterm babies (Chauhan et al., 2022). C. sakazakii outbreak in infant formula was long reported in England, 1958, after which relevant outbreaks were reported globally every few years (Henry & Fouladkhah, 2019). Just recently in 2022, outbreaks of C. sakazakii were reported in the US, which affected over 40% of infant formula sold in the US, leading to a massive shortage of formula over 6 months (Goodman, 2022). Despite the risk of C. sakazakii has long been recognized, regular sanitization approaches in the dairy industry might be insuffcient for complete sterilization since they have strong resistance to hostile stress, such as high acid, high temperature, and low water activity (Chen et al., 2019; Henry & Fouladkhah, 2019; Ueda, 2017), which could still pose a considerable residual hazard. The commonly used sanitizing approach in the dairy industry are chlorine, quaternary ammonium chloride (QAC), and hot water saniti- zation, due to their remarkable benefts (e.g., good effectiveness, ease of usage, and cheap cost) (Panebianco et al., 2022; Tan et al., 2022). Aside from that, organic acids are gaining increasing attention in sanitizing procedures owing to their antibacterial properties as well as being generally regarded as safe (GRAS) (Chauhan et al., 2022). Moreover, * Corresponding author. E-mail address: e0669601@u.nus.edu (Z. Lin). Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres https://doi.org/10.1016/j.foodres.2023.113162 Received 18 January 2023; Received in revised form 14 June 2023; Accepted 16 June 2023