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Robert H. Smith Faculty of Agriculture, Food and Environment,
The Hebrew University of Jerusalem, 
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Shumeiko, V. ; Paltiel, Y. ; Bisker, G. ; Hayouka, Z. ; Shoseyov, O. A nanoscale paper-based near-infrared optical nose (NIRON). Biosensors and Bioelectronics 2021, 172, 112763. Publisher's VersionAbstract
Electronic noses (e-nose) and optical noses (o-nose) are two emerging approaches for the development of artificial olfactory systems for flavor and smell evaluation. The current work leverages the unique optical properties of semiconducting single-wall carbon nanotubes (SWCNTs) to develop a prototype of a novel paper-based near-infrared optical nose (NIRON). We have drop-dried an array of SWCNTs encapsulated with a wide variety of peptides on a paper substrate and continuously imaged the emitted SWCNTs fluorescence using a CMOS camera. Odors and different volatile molecules were passed above the array in a flow chamber, resulting in unique modulation patterns of the SWCNT photoluminescence (PL). Quartz crystal microbalance (QCM) measurements performed in parallel confirmed the direct binding between the vapor molecules and the peptide-SWCNTs. PL levels measured before and during exposure demonstrate distinct responses to the four tested alcoholic vapors (ethanol, methanol, propanol, and isopropanol). In addition, machine learning tools directly applied to the fluorescence images allow us to distinguish between the aromas of red wine, beer, and vodka. Further, we show that the developed sensor can detect limonene, undecanal, and geraniol vapors, and differentiate between their smells utilizing the PL response pattern. This novel paper-based optical biosensor provides data in real-time, and is recoverable and suitable for working at room temperature and in a wide range of humidity levels. This platform opens new avenues for real-time sensing of volatile chemical compounds, odors, and flavors.
Yehuda, A. ; Slamti, L. ; Malach, E. ; Lereclus, D. ; Hayouka, Z. Elucidating the Hot Spot Residues of Quorum Sensing Peptidic Autoinducer PapR by Multiple Amino Acid Replacements. Front Microbiol 2019, 10, 1246.Abstract
The quorum sensing (QS) system of , an opportunistic human pathogen, utilizes the autoinducing PapR peptide signal that mediates the activation of the pleiotropic virulence regulator PlcR. A set of synthetic 7-mer PapR-derived peptides (PapR; ADLPFEF) have been shown to inhibit efficiently the PlcR regulon activity and the production of virulence factors, reflected by a loss in hemolytic activity without affecting bacterial growth. Interestingly, these first potent synthetic inhibitors involved D-amino acid or alanine replacements of three amino acids; proline, glutamic acid, and phenylalanine of the heptapeptide PapR. To better understand the role of these three positions in PlcR activity, we report herein the second generation design, synthesis, and characterization of PapR-derived combinations, alternate double and triple alanine and D-amino acids replacement at these positions. Our findings generate a new set of non-native PapR-derived peptides that inhibit the PlcR regulon activity and the production of virulence factors. Using the amino acids substitution strategy, we revealed the role of proline and glutamic acid on PlcR regulon activation. Moreover, we demonstrated that the D-Glutamic acid substitution was crucial for the design of stronger PlcR antagonists. These peptides represent potent synthetic inhibitors of QS and constitute new and readily accessible chemical tools for the study of the PlcR system. Our method might be applied to other quorum sensing systems to design new anti-virulence agents.
Amso, Z. ; Hayouka, Z. Antimicrobial random peptide cocktails: a new approach to fight pathogenic bacteria. Chem Commun (Camb) 2019, 55, 2007-2014.Abstract
Antibiotic resistance in bacteria has become a serious threat to public health, and therefore there is an urgent need to develop new classes of antimicrobial agents. Nowadays, natural antimicrobial peptides (AMPs) and their synthetic derivatives are considered as promising alternatives to traditional antibiotics. The broad molecular diversity of AMPs, in terms of sequences and structures, suggests that their activity does not depend on specific features of amino acid sequence or peptide conformation. We therefore selected two common properties of AMPs, (high percentage of hydrophobic and cationic amino acids), to develop a novel approach to synthesize random antimicrobial peptide mixtures (RPMs). Instead of incorporating a single amino acid at each coupling step, a mixture of hydrophobic and cationic amino acids in a defined proportion is coupled. This results in a mixture that contains up to 2n sequences, where n is the number of the coupling step, of random peptides with a defined composition, stereochemistry, and controlled chain length. We have discovered that RPMs of hydrophobic and cationic α-amino acids, such as phenylalanine and lysine, display strong and broad antimicrobial activity towards Gram-negative, Gram-positive, clinically isolated antibiotic resistant "superbugs", and several plant pathogenic bacteria. This review summarizes our efforts to explore the mode of action of RPMs and their potential as bioactive agents for multiple applications, including the prevention of biofilm formation and degradation of mature biofilm (related to human health), reduction of disease severity in plant bacterial disease models (related to crop protection), and inhibition of bacterial growth in milk (related to food preservation). All our findings illustrate the effectiveness of RPMs and their great potential for various applications.
Stern Bauer, T. ; Menagen, B. ; Avnir, D. ; Hayouka, Z. Random peptide mixtures entrapped within a copper-cuprite matrix: new antimicrobial agent against methicillin-resistant Staphylococcus aureus. Sci Rep 2019, 9 11215.Abstract
The emergence of global antibiotic resistance necessitates the urgent need to develop new and effective antimicrobial agents. Combination of two antimicrobial agents can potentially improve antimicrobial potency and mitigate the development of resistance. Therefore, we have utilized metal molecular doping methodology whereby antimicrobial random peptides mixture (RPMs) are entrapped in a bactericidal copper metal matrix. The copper/RPM composite exhibits greater antimicrobial activity toward methicillin-resistant Staphylococcus aureus (MRSA) than either copper or RPMs alone. Our findings indicate that this bactericidal antimicrobial biomaterial could be utilized to efficiently eradicate antibiotic-resistant pathogenic bacteria for health, agricultural and environmental applications.
Ghate, V. ; Zelinger, E. ; Shoyhet, H. ; Hayouka, Z. Inactivation of Listeria monocytogenes on paperboard, a food packaging material, using 410 nm light emitting diodes. Food Control 2019, 96, 281 - 290. Publisher's VersionAbstract
Light emitting diodes of wavelength 410 nm were used to inactivate Listeria monocytogenes stains on paperboard, an increasingly popular food packaging material. The integrity of the cell membranes was examined using differential fluorescent staining. Scanning electron microscopy (SEM) was used to obtain a deeper understanding of L. monocytogenes stain formation on paperboard and the damage caused to the cells by the LEDs. While the planktonic L. monocytogenes population could be completely inactivated following a brief lag phase that lasted about 20 min, the illumination of the sessile population left some persisters despite immediate commencement of the inactivation. Planktonic populations of inocula sized 3, 5 and 7 log CFU/mL were reduced below the detection limit in 54, 80 and 84 min respectively, whereas it took 120 and 390 min to reach constancy in the sessile populations of inocula sized 5 and 7 log CFU/cm2. The number of membrane-damaged cells was seen to increase with the illumination time. SEM images provided evidence of the protection conferred by the stain on the underlying cells. This study demonstrates that blue LEDs have the potential to reduce the risk of L. monocytogenes contamination from paperboard cartons under refrigeration.
Ocłoń, E. ; Solomon, G. ; Hrabia, A. ; Druyan, S. ; Hayouka, Z. ; Gertler, A. New reagents for poultry research: preparation, purification, and in vitro evaluation of non-PEGylated and mono-PEGylated chicken prolactin. Poultry Science 2018, 97, 3277 - 3285. Publisher's VersionAbstract
ABSTRACT Recombinant chicken prolactin (chPRL), expressed in Escherichia coli and purified as a monomer, was successfully PEGylated and purified to homogeneity as a mono-PEGylated protein (PEG-chPRL). Its biological activity was estimated by its ability to interact with human prolactin receptor extracellular domain (hPRLR-ECD) and stimulate PRLR-mediated proliferation in Nb2-11C cells. PEG-chPRL activity in a cell bioassay was 10-fold lower than that of non-PEGylated chPRL, but only 2-fold lower in a binding assay to hPRLR-ECD. The CD spectra of non-PEGylated and PEGylated chPRL were almost identical and similar to that of hPRL, indicating proper refolding. Although the PEGylation of chPRL resulted in lower activity in vitro, PEG-chPRL was absorbed more slowly than chPRL, remained in the circulation 16 h longer. Furthermore the effects of PEG-chPRL injections in chickens on subsequent corticosteroid levels in blood were significantly profound compared to chPRL. These favorable PEGylation-induced pharmacokinetic alterations should improve efficacy of PEG-chPRL in in vivo experiments, as dosing frequency can be reduced due to its prolonged persistence in the circulation, and thus reduce the frequency of dosing. Furthermore, hydrophobic interaction chromatography was successfully adopted to isolate PEG-chPRL as a better alternative for separation of PEGylated PRL, and is likely to be successfully applicable to other proteins.
Oclon, E. ; Solomon, G. ; Hayouka, Z. ; Salame, T. M. ; Goffin, V. ; Gertler, A. Novel reagents for human prolactin research: large-scale preparation and characterization of prolactin receptor extracellular domain, non-pegylated and pegylated prolactin and prolactin receptor antagonist. Protein Eng Des Sel 2018, 31, 7-16.Abstract
To provide new tools for in vitro and in vivo prolactin (PRL) research, novel protocols for large-scale preparation of untagged human PRL (hPRL), a hPRL antagonist (del 1-9-G129R hPRL) that acts as a pure antagonist of hPRL in binding to hPRL receptor extracellular domain (hPRLR-ECD), and hPRLR-ECD are demonstrated. The interaction of del 1-9-G129R hPRL with hPRLR-ECD was demonstrated by competitive non-radioactive binding assay using biotinylated hPRL as the ligand and hPRLR-ECD as the receptor, by formation of stable 1:1 complexes with hPRLR-ECD under non-denaturing conditions using size-exclusion chromatography, and by surface plasmon resonance methodology. In all three types of experiments, the interaction of del 1-9-G129R hPRL was equal to that of unmodified hPRL. Del 1-9-G129R hPRL inhibited the hPRL-induced proliferation of Baf/LP cells stably expressing hPRLR. Overall, the biological properties of del 1-9-G129R hPRL prepared by the protocol described herein were similar to those of the antagonist prepared using the protocol reported in the original study; however, the newly described protocol improved yields by >6-fold. To provide long-lasting hPRL as a new reagent needed for in vivo experiments, we prepared its mono-pegylated analogue and found that pegylation lowers its biological activity in a homologous in vitro assay. As its future use will require the development of a PRL antagonist with highly elevated affinity, del 1-9-G129R hPRL was expressed on the surface of yeast cells. It retained its binding capacity for hPRLR-ECD, and this methodology was shown to be suitable for future development of high-affinity hPRL antagonists using a library of randomly mutated open reading frame of del 1-9-G129R hPRL and selecting high-affinity mutants by yeast surface display methodology.
Hu, S. ; Zhao, Y. ; Hayouka, Z. ; Wang, D. ; Jiao, S. Inactivation kinetics for Salmonella typhimurium in red pepper powders treated by radio frequency heating. Food Control 2018, 85, 437 - 442. Publisher's VersionAbstract
Radio frequency (RF) dielectric heating has been investigated to inactivate pathogens in some low-moisture foods. This study was aimed to evaluate RF inactivation effects on Salmonella typhimurium in red pepper powders, by focusing on the influence of sample initial water activity (aw) and applying Weibull model to describe the inactivation curves. The experimental results showed that RF heating rate increased when aw was in the range of 0.57–0.71, but decreased after aw reached to 0.71. During the come-up time of RF heating, 2–3 log reduction of the pathogen was achieved depending on aw levels. Increasing initial aw could first increased log reductions and then decreased the log reductions, optimum aw level was 0.71 for RF inactivation of Salmonella in red pepper powders. For red pepper powders with aw of 0.71, RF heating to 70 °C (come-up time was 110 s) with holding time over 60 s could achieve >5 log reduction of S. typhimurium. Weibull model well fitted the survival curves of the pathogen with goodness of fit (R2 > 0.93, RMSE<0.29). Scale factor (b) of the model increased with treatment temperature increasing, while the shape factor (n) was independent on temperature. This study provided basic guideline for using RF heating to inactivate Salmonella in red pepper powders.
Topman, S. ; Tamir-Ariel, D. ; Bochnic-Tamir, H. ; Stern Bauer, T. ; Shafir, S. ; Burdman, S. ; Hayouka, Z. Random peptide mixtures as new crop protection agents. Microb Biotechnol 2018, 11, 1027-1036.Abstract
Many types of crops are severely affected by at least one important bacterial disease. Chemical control of bacterial plant diseases in the field vastly relies on copper-based bactericides, yet with limited efficacy. In this study, we explored the potential of two random peptide mixture (RPM) models as novel crop protection agents. These unique peptide mixtures consist of random combination of l-phenylalanine and l- or d-lysine (FK-20 and FdK-20, respectively) along the 20 mer chain length of the peptides. Both RPMs displayed powerful bacteriostatic and bactericidal activities towards strains belonging to several plant pathogenic bacterial genera, for example, Xanthomonas, Clavibacter and Pseudomonas. In planta studies in the glasshouse revealed that RPMs significantly reduced disease severity of tomato and kohlrabi plants infected with Xanthomonas perforans and Xanthomonas campestris pv. campestris respectively. Moreover, RPM effects on reduction in disease severity were similar to those exerted by the commercial copper-based bactericide Kocide 2000 that was applied at a 12-fold higher concentration of the active compound relative to the RPM treatments. Importantly, the two tested RPM compounds had no toxic effect on survival of bees and Caco-2 mammalian cells. This study demonstrates the potential of these innovative RPMs to serve as crop protection agents against crop diseases caused by phytopathogenic bacteria.
Stern Bauer, T. ; Hayouka, Z. Random mixtures of antimicrobial peptides inhibit bacteria associated with pasteurized bovine milk. J Pept Sci 2018, 24, e3088.Abstract
The shelf life of pasteurized bovine milk is limited by microorganism activity as surviving bacteria continue to grow in the bovine milk, eventually causing milk spoilage. In the current study, we used matrix-assisted laser desorption ionization time of flight mass spectrometry to identify pasteurized bovine milk-associated mesophilic and psychrotrophic bacteria. We have recently designed random cationic peptide mixtures that possess strong antimicrobial and antibiofilm properties. These compounds are cheap and easy to synthesize and represent a new class of antimicrobial agents. Here, we show that the random peptide mixtures are able to efficiently eradicate the bacteria identified as associated with pasteurized bovine milk, and reduced significantly the growth of Bacillus subtilis in milk. We propose these antimicrobial peptides as potential candidates for integration in bioactive milk and food packaging to prevent bacterial growth and extend the shelf life of food.
Ocłoń, E. ; Solomon, G. ; Hayouka, Z. ; Gertler, A. Preparation of biologically active monomeric recombinant zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) recombinant growth hormones. Fish Physiology and Biochemistry 2018, 44, 1215 - 1222. Publisher's VersionAbstract
Fish growth hormones (GHs) play an important role in regulating growth, metabolism, reproduction, osmoregulation, and immunity and have thus garnered attention for their application in aquaculture. Zebrafish GH (zGH) cDNA or rainbow trout GH (rtGH) cDNA was cloned into the pMon3401 vector, expressed in MON105-competent Escherichia coli and purified to homogeneity. Their biological activity was evidenced by their ability to interact with ovine GH receptor extracellular domain and stimulate GH receptor-mediated proliferation in FDC-P1-3B9 cells stably transfected with rabbit GH receptor. The relative affinity of zGH and rtGH, estimated by IC50, was about 38-fold and 512-fold lower, respectively, than ovine GH. This is likely the reason for the low biological activity in cells with rabbit GH receptor, ~ 36-fold lower for zGH and ~ 107-fold lower for rtGH than for human GH. This was not due to improper refolding, as evidenced by circular dichroism analysis. Predicting the activity of fish GHs is problematic as there is no one single optimal in vitro bioassay; heterologous assays may be ambiguous, and only homologous assays are suitable for measuring activity.
Yehuda, A. ; Slamti, L. ; Bochnik-Tamir, R. ; Malach, E. ; Lereclus, D. ; Hayouka, Z. Turning off Bacillus cereus quorum sensing system with peptidic analogs. Chemical Communications 2018, 54, 9777 - 9780. Publisher's VersionAbstract
We explored quenching of the PlcR–PapR quorum-sensing system in Bacillus cereus. We generated PapR7-peptidic derivatives that inhibit this system and thus the production of virulence factors, reflected by a loss in hemolytic activity, without affecting bacterial growth. To our knowledge, these peptides represent the first potent synthetic inhibitors of quorum-sensing in B. cereus.
Vilela, C. ; Kurek, M. ; Hayouka, Z. ; Röcker, B. ; Yildirim, S. ; Antunes, M. D. C. ; Nilsen-Nygaard, J. ; Pettersen, M. K. ; Freire, C. S. R. A concise guide to active agents for active food packaging. Trends in Food Science & Technology 2018, 80, 212 - 222. Publisher's VersionAbstract
BackgroundThe ever-growing world population results in the ineluctable increase of food demand which translates in the augment of the global market of packaging materials. Hence, the concept of active packaging materializes as a technology to enhance the safety, quality and shelf-life of the packaged foods. Active packaging systems can contribute to the reduction of food waste by providing, apart from an inert barrier to external conditions, several functions associated with food preservation, namely absorbing/scavenging, releasing/emitting and removing properties, temperature, microbial and quality control. Scope and approach The purpose of this review is to present a concise (but wide-ranging) appraisal on the latest advances in active agents for active food packaging. Emphasis is placed on active functions such as antimicrobial and antioxidant activity, oxygen and ethylene scavenging, and carbon dioxide emitting. An effort was made to highlight representative articles that prompted research on active agents towards viable market solutions. Key findings and conclusions Active packaging is a thriving field given its duality as barrier to external detrimental factors and active role in food preservation and quality. The use of natural active agents is a flourishing field due to the general concern towards natural-based additives. Nevertheless, research is still in its early stages with a long way to go in the design of innovative and economical active packaging materials containing appropriate active agents. The interaction between packaging, environment and food is the key challenge for achieving commercial translation.
Hayouka, Z. ; Bella, A. ; Stern, T. ; Ray, S. ; Jiang, H. ; Grovenor, C. R. M. ; Ryadnov, M. G. Binary Encoding of Random Peptide Sequences for Selective and Differential Antimicrobial Mechanisms. Angewandte Chemie International Edition 2017, 56, 8099-8103. Publisher's VersionAbstract
Abstract Binary encoding of peptide sequences into differential antimicrobial mechanisms is reported. Such sequences are random in composition, but controllable in chain length, are assembled from the same two amino acids, but differ in the stereochemistry of one. Regardless of chirality, the sequences lyse bacteria including the “superbugs” methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Sequences with the same chirality, so-called homochiral sequences, assemble into antimicrobial pores and form contiguous helices that are biologically promiscuous and hemolytic. By contrast, heterochiral sequences that lack such persistence selectively attack bacterial membranes without oligomerizing into visible pores. These results offer a mechanistic rationale for designing membrane-selective and sequence-independent antimicrobials.
Stern, T. ; Zelinger, E. ; Hayouka, Z. Random peptide mixtures inhibit and eradicate methicillin-resistant: Staphylococcus aureus biofilms. Chemical Communications 2016, 52, 7102 - 7105. Publisher's Version