Antimicrobial isothiocyanates from Brassicaceae glucosinolates Glc S R S δ+ Antimicrobial isothiocyanates Myr, pH 5-7 S C H2O R C R' SH R C R' N Glc N S N SO3 H + O SO 2- H from Brassicaceae glucosinolates 4 Analysis, reactivity, quantitative structure-activity relationships O O N S N S C C S 2 S 8 O O N S N S C C S S 7 N S O C S 2 N S C S 9 N C S O N S C S 7 O O N S C O Silvia Andini 2020 S S C N 2 S C N Silvia Andini Propositions 1. Isothiocyanates (ITCs) with methylsulfinyl or methylsulfonyl groups represent ITCs with a good antimicrobial activity (MIC ≤ 25 µg/mL). (this thesis) 2. Total amino acid content of growth media influences the antimicrobial activity of ITCs. (this thesis) 3. The saying “eat your breakfast like an empress, lunch like a queen, and dine like a pauper” only applies for obese women to lose body weight. (Key refs: Jakubowicz et al. (2013) Obesity 21, 2504- 2512; Andersen et al. (2004) Int. J. Obes. 28, 1338-1343) 4. Agroforestry with a financial compensation is a solution to reduce deforestation in Indonesia. 5. Dutch colonization of Indonesia has caused a false perception of typical Indonesian foods, including sate ajam, ketjap manis, and sambal oelek. 6. Social media addiction should be viewed as an illness and requires an appropriate rehabilitation treatment. Propositions belonging to the thesis entitled: Antimicrobial isothiocyanates from Brassicaceae glucosinolates Analysis, reactivity, and quantitative structure-activity relationships Silvia Andini Wageningen, 06 November 2020 Antimicrobial isothiocyanates from Brassicaceae glucosinolates Analysis, reactivity, and quantitative structure- activity relationships Silvia Andini Thesis committee Promotor Prof. Dr J.-P. Vincken Professor of Food Chemistry Wageningen University & Research Co-promotor Dr C. Araya-Cloutier Assistant professor, Laboratory of Food Chemistry Wageningen University & Research Other members Prof. Dr V. Fogliano, Wageningen University & Research Dr N. Agerbirk, University of Copenhagen, Denmark Dr J.-W. Sanders, Unilever R&D, Wageningen Dr A.B.G. Bonnema, Wageningen University & Research This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Antimicrobial isothiocyanates from Brassicaceae glucosinolates Analysis, reactivity, and quantitative structure- activity relationships Silvia Andini Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus, Prof. Dr A.P.J. Mol, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 6 November 2020 at 1:30 pm in the Aula. Silvia Andini Antimicrobial isothiocyanates from Brassicaceae glucosinolates Analysis, reactivity, and quantitative structure-activity relationships 208 pages. PhD thesis, Wageningen University, Wageningen, The Netherlands (2020) With references, with summary in English ISBN 978-94-6395-454-9 DOI https:/doi.org/10.18174/526437 Abstract Isothiocyanates (ITCs) are electrophilic phytochemicals naturally originating from glucosinolates (GSLs), the major phytochemicals in the plant family of Brassicaceae. ITCs are released when GSLs come in contact with myrosinase. This is part of the Brassicaceae defence system against their natural enemies. The aim of this thesis was to explore the potential of ITCs as natural antimicrobials, e.g. as food preservatives. For this, their production, analysis, reactivity, and (quantitative) structure-activity relationships were studied. Fungal elicitation was studied as an attempt to increase content and structural diversity of GSLs. (Non-)pathogenic fungal elicitation, during Brassicaceae seed germination, did not increase structural diversity nor content of GSLs further than what could be obtained by germination alone. Meanwhile, non-elicited seedlings contained higher or equal GSL content compared to the untreated seeds. As GSLs were present in high abundance in Brassicaceae seeds, the seeds were employed as the source of antimicrobial ITCs via extraction of GSLs, followed by a myrosinase treatment. For this, a method to analyze GSLs and ITCs simultaneously using RP-UHPLC-ESI-MSn was developed and validated with 14 GSLs and 15 ITCs, consisting of 8 subclasses differing in side chain configuration. The method enabled monitoring the in vitro enzymatic conversion of GSLs to ITCs. Due to their electrophilicity, antimicrobial activity of ITCs was negatively affected by nucleophile richness of growth media. In nucleophile-poor growth media, antimicrobial activity of ITCs, particularly x-(methylsulfinyl)alkyl ITC (MSITC) and x-(methylsulfonyl)alkyl ITC (MSoITC), was remarkably improved by a factor of at least 4, compared to that in the nucleophile-rich. Of 9 ITC subclasses tested, MSITC and MSoITC were the most promising ones. Antimicrobial activity of MSITC and MSoITC was dependent on chain length. The short-chained MSITC and MSoITC had good antimicrobial activity (minimum inhibitory concentration, MIC ≤ 25 µg/mL) against Gram– bacteria and Gram+ bacteria, whereas the long-chained ones had good antimicrobial activity against Gram+ bacteria and fungi. A QSAR study revealed that partial charge, polarity, reactivity, and shape were the main physicochemical properties defining the antimicrobial activity of ITCs. The 2 developed QSAR models had good internal prediction power (Q adj > 0.80) and successfully predicted activity of ITC-rich Brassicaceae extracts. Table of contents Chapter 1 General introduction 1 Chapter 2 Modulation of glucosinolate composition in 27 Brassicaceae seeds by germination and fungal elicitation Chapter 3 Simultaneous analysis of glucosinolates and 71 isothiocyanates by RP-UHPLC-ESI-MSn Chapter 4 The interplay between antimicrobial activity and 103 reactivity of isothiocyanates Chapter 5 QSAR-based physicochemical properties of 123 isothiocyanate antimicrobials against Gram- negative and Gram-positive bacteria Chapter 6 General discussion 167 Summary 197 Acknowledgements 201 About the author 205 CHAPTER 1 General Introduction There is an urgent call to continuously discover new antimicrobial compounds, especially derived from nature. Plant secondary metabolites, so-called phytochemicals, involved in plant defense, potentially are new natural antimicrobial candidates, which can be useful to healthcare systems and food industries, as well as other industries. Isothiocyanates (ITCs) are typical phytochemicals involved in the defense system of the Brassicaceae family, a widely cultivated and economically important plant family. ITCs are released upon degradation of glucosinolates (GSLs) by the activity of myrosinase. ITCs, likewise GSLs, are structurally diverse. Reports on antimicrobial activity of ITCs and their potential application are limited to just a few ITCs. Considering the large structural diversity of ITCs, systematic investigations of structure-activity relationship of ITCs as antimicrobials are scarce. Furthermore, ITCs are electrophilic making them reactive towards nucleophiles, which are abundantly present in many food products and microbial growth media. The consequences of this have been relatively underexposed in scientific literature. This chapter addresses (i) the need for new natural antimicrobial compounds, (ii) structural diversity of GSLs and ITCs, including their analysis, (iii) occurrence of GSLs and ITCs, (iv) approaches to modulate structural diversity and content of GSLs in Brassicaceae seedlings, and (v) ITCs as potential natural antimicrobial candidates, also considering their reactivity. Chapter 1 1.1. The need for new natural antimicrobial compounds Food industries are facing a continuous challenge to develop clean label and mildly processed, as well as safe and convenient food products, to meet consumer demands.1-3 Furthermore, secondary shelf life of food products is an underestimated issue and receives less attention than primary shelf life.4 Primary shelf life, often referred to as just shelf life, is the length of time after production and packaging during which a food product in the enclosed state stays at the required level of quality and safety under a well-defined storage condition. Secondary shelf life, so-called period after opening or open shelf life, is the length of time after opening of a food product during which it may be consumed at an acceptable quality and safety level. Released from manufacturers, food products should be microbiologically safe with zero or very low microbial contamination. Once opened, food products acquire microbial contamination from the environment and upon handling by consumers. Vinegar, sugar, and salt are often added to food products, not only for taste and flavor, but also for extending the secondary shelf life. Alternative ingredients for extending secondary shelf life of various food products (heated and raw) without influencing their sensory attributes are of great interest. At the same time the world is facing the problem of antimicrobial resistance (AMR) and persistence causing the treatment of microbial infections to become more difficult, with a concomitant increase in the number of casualties.5 In 2016, 700,000 deaths globally caused by AMR infections were estimated.6 This number is predicted to grow to 10 million in 2050, along with a global economic cost of 100 trillion USD, if no action is taken to tackle the global AMR problem.6 Altogether, there is an urgent call to continuously discover new antimicrobial