Valorization of licorice root by-product for safe, natural antimicrobials
Plant antimicrobials offer high effectiveness, low toxicity and low incidence of resistance development.
Therefore, industrial interest in developing products containing such compounds to combat (persistent) microorganisms in different settings is growing. In addition, consumers are demanding more naturally preserved (food) products, and they strongly oppose use of antibiotics in meat production or synthetic/artificial preservatives (e.g. benzoates). Licorice roots have a longstanding safe use in traditional medicine, and their prenylated phenolic compounds (e.g. glabridin) were
shown to have antimicrobial activity with potency similar to commonly used antibiotics. In current industrial applications of licorice roots, these antimicrobial phenolics are left untouched, as part of the licorice root by-product (spent).
This project seeks the development of broad spectrum antimicrobial extracts by (i) identifying the most potent antimicrobials from licorice root spent and their spectrum of activity against problematic spoilage and pathogenic microorganisms relevant to food, feed, and oral care; (ii) assessing the safety and potential (positive/negative) health aspects of licorice root spent antimicrobials, by using an innovative 2D mini-gut organoid model; (iii) defining routes for valorization of licorice root spent from commercially relevant licorice species.
MMIPs mission is to obtain a safer and more sustainable food chain by 2030, and aims to reduce energy and water use, in combination with reduction of food waste. Regarding that last aspect, especially optimization of waste streams is vital. In LicoRoot, we specifically target reduction of food waste, by valorizing the waste stream of “licorice root extract”, which is commercially used in e.g. the confectionary and tobacco industry. Furthermore, LicoRoot will deliver safe natural antimicrobials to be used in food, feed and oral care applications. This will allow these sectors to reduce the amount of traditional antimicrobials (e.g. antibiotics, preservatives) used, which will help improve health and reduce the antimicrobial resistance problem faced worldwide.
One of the priorities of MMIP includes improving resource efficiency and flexibility, such as the use waste streams. LicoRoot specifically addresses this priority by valorizing waste stream of “licorice root extract”.
WP1: Purification and chemical characterization of Glycyrrhiza glabra, G. inflata, and G. uralensis spent extracts
- Supply of spent from G. glabra, G. inflata and G. uralensis (Ruitenberg (RU)) and chemical characterization (Laboratory of Food Chemistry (WU-FCH)), including:
o General quality control (RU).
o Ochratoxin contaminant content (RU).
o Glycyrrhizin content (RU): % of glycyrrhizin in spent should be 80% will be considered for the structure-activity relationships studies.
WP2: Antimicrobial profiling, Structure Activity Relationship and interaction with food ingredients
- High-throughput antimicrobial screening of EtOAc extracts and fractions. Assays include:
o Antimicrobial susceptibility assay using broth micro-dilution (WU-FCH, Unilever (UL), Delacon (DE), Cavex (CA)): extracts with minimum inhibitory concentration (MIC) below 5 mg/mL and fractions with a MIC below 1.0 mg/mL against the target microorganisms will be consider promising.
o Spectrum of activity: testing against different types of microorganisms. Extracts or fractions that show a broad spectrum of activity (bacteria, yeast, moulds) will be considered promising.
o Evaluation of ingredient interactions with extracts and fractions enriched in prenylated phenolic compounds (UL): antimicrobial activity will be measured in the presence of ingredients (e.g. oil, proteins) based on the partners product recipes.
o Oral care performance tests (CA): the top 2-3 extracts and fractions enriched in prenylated phenolic compounds that show promising MIC values against bacteria relevant to oral care, and that comply with safety criteria, will be tested in small-scale dental practice performance assays representative for oral care products (toothpaste and gels).
Year 3 and 4:
- Antimicrobial profiling of purified compounds and structure-activity relationships. Assays (may) include:
o Antimicrobial susceptibility assay using broth micro-dilution (WU-FCH): compounds with minimum inhibitory concentration (MIC) below 0.2 mg/mL against the target microorganisms will be considered promising for partners applications.
o Mechanism of action of promising molecules will be determined using spectrophotometric, microscopic, and fluorescence imaging techniques (WU-FCH).
o Biofilm formation inhibition and expression of quorum-sensing genes (DE).
o Antimicrobial activity will be measured in the presence of ingredients (e.g. oil, proteins) based on the partners product recipes (UL)
WP 3: Safety and health aspects of G. glabra, G. inflata, and G. uralensis extracts and pure prenylated phenolic compounds
- Safety profiling of top 5 candidate extracts or fractions will be performed using an organoid system (WU-HNE). A human organoid model system is under development in a currently running project at WU-HNE, and it might already be available at the intermediate stages of this TKI project. When fully operational, the human organoid model system will replace that of mouse. The organoid system will be used to measure:
o Toxicity in organoids using an LDH assay: candidate extracts or fractions will be tested at concentrations where antimicrobial activity was observed (e.g. 0.5MIC, MIC, 2MIC). Samples, which are found to be toxic at their MIC value, will be discarded from the study.
- Effects on intestinal barrier function (WU-HNE): Those samples that passed the toxicity criteria, will be subjected to the organoid model system. The following analysis will be done:
o Membrane permeability using TEER and FITC-dextran assays.
o Mucin-2 (mRNA, histological analysis (Alcian blue/PAS staining), and immune-histochemical Muc-2 detection), claudin, and cytokines (mRNA by qPCR).
o Pro-inflammatory cytokine production by ELISA.
Compounds with no detrimental effects or, in contrast, positive effects on gut barrier function will be considered for further study.
Year 3 and 4:
- Safety profiling of top 2-3 candidate purified compounds (WU-HNE), by measuring:
o Toxicity using the LDH assay: candidate compounds will be tested at concentrations where antimicrobial activity was observed (e.g. 0.5MIC, MIC, 2MIC). Samples, which are found to be toxic at their MIC value, will be discarded from the study.
- Effects on intestinal barrier function (WU-HNE): those purified compounds, that passed the toxicity assay criteria, will be tested with the organoid model system for:
o Barrier function.
o Whole genome transcriptome analysis and GSEA.
- Regulatory aspects of top candidate products (extracts, fractions or purified compounds) (all partners): for those samples that showed promising antimicrobial and safety profiles, industrial partners will investigate the regulatory aspect for the utilization of such samples in their specific applications.
WP 4: Writing thesis and publications