Octyl Gallate Use to Protect Seeds from Foodborne Fungal Pathogens ^†

Abstract

There is limited efficacy with conventional seed sanitation methods, directly affecting food safety. Insufficient elimination of mycotoxin-producing fungi contaminating seed surfaces can result in high mycotoxin contamination. In this study, a new seed sanitation formula was investigated by examining molecules repurposed from the United States Food and Drug Administration (FDA)-approved food additives as active ingredients. The selected benzaldehyde analog, octyl gallate (OG; octyl 3,4,5-trihydroxybenzoic acid), previously shown to inhibit mycotoxin production, could function as heat-sensitizing agents when co-applied with mild heat. The co-application substantially enhanced the sanitation efficacy against fungi contaminating crop seeds, whereas the seed germination rate was unaffected. Therefore, OG-based heat sensitization could be a promising tool to achieve safe and cost-effective pathogen control in agriculture/food production.

Contamination of crops by pathogens, especially those resistant to conventional biocides, represents a major food safety and security concern [1]. Moreover, outbreaks of commodity-specific food sources, such as crop contamination by fungi-produced hepato-carcinogenic mycotoxins, directly affect the health and safety of the public.

Seed treatment enables healthy crop establishment in the fields through better seed preservation and crop protection against a wide variety of pathogens [2,3]. Compared to other protection measures, seed treatment can achieve a low-cost seed/crop protection since the treatment requires a relatively small amount of active ingredients, thus allowing sustainable disease control such as the prevention of mycotoxin contamination at the earlier stage of crop growth. The development of novel seed-treatment formulations that can be applied as alternatives to the conventional toxic chemical compositions will also facilitate sustainable food production.

Heat treatment became one of the important strategies for the prevention of pathogen contamination in agricultural or food production [4]. While heat treatment can ensure the reduction of pathogen contamination or postharvest decay, intensive heat treatment can result in the deterioration of the quality of the crops or food products. Hurdle technology is an approach where the combined application of different types of preservation methods at reduced individual intensities could achieve increases in the effectiveness of antimicrobial treatments [5].

Drug or compound repurposing is the process where already marketed drugs or compounds which were previously applied for treating human diseases, foods, etc., are repurposed for the control of microbial pathogens, weeds, or other pests [6]. The main merit of drug or compound repurposing is that the mode of action, molecular targets, or safety of the commercial drugs/compounds have already been characterized. Considering the identification and development of entirely new anti-pest substances is an expensive and time-consuming process, the alternative approach, viz., drug or compound repurposing, has been recently investigated [7]. We investigated the heat-sensitizing capability of a new seed sanitation formula as a new hurdle technology by using molecules repurposed from commercial food additives.

Antimicrobial assay: The heat-sensitizing effect of benzaldehyde analogs, currently used as food additives/derivatives [8], was examined using the mycotoxin-producing fungus Aspergillus flavus. Test samples were treated with mild heat (57.5 °C) or maintained at room temperature (RT; 22.0 °C), then entire samples were cultured for 48 h at 35 °C onto the recovery agar (potato dextrose agar (PDA)) plates. Then, the most effective benzaldehyde analog, octyl gallate (OG; octyl 3,4,5-trihydroxybenzoic acid) (Figure 1, structure), was examined further at 0.1 to 0.3 mM to determine the optimum treatment condition (mild heat (57.5 °C) or RT (22.0 °C)).

Antifungal seed disinfection assay: Effects of OG plus mild heat (hurdle technology) on seed sanitation was performed on Brassica rapa (cabbage) seeds. For seed treatments, the co-application of OG (3 mM) and mild heat (50 °C) (20 to 30 min) was investigated on A. flavus-contaminated seeds. The germination of seeds and fungal growth on the surfaces of germinated seeds were monitored for 7 days. Statistical analysis (student’s t-test) was performed according to “Statistics to use” [9], where a p < 0.05 was considered significant.

As shown in Figure 2, co-treatment with the OG at 3 mM and mild heat (50 °C) for 20 min completely inhibited the growth of A. flavus, while the germination frequency of the crop seeds was not affected when compared to the control. Either 2 mM or 4 mM OG treatments were less effective compared to the 3 mM treatment, namely, fungal contamination was observed at 2 mM or less seed germination at 4 mM of OG, respectively.

In summary, the new utility of repurposed OG as a heat-sensitizing agent has been identified; OG exhibited potent heat-sensitizing capability. Thus, results from this study provide means that can enhance the capacity of accepted intervention strategies, such as pasteurization/heat treatment, or alternatives to toxic antifungal agents, such as the seed disinfection agents Thiram, Ferbam, and Ziram; the Pest Management Regulatory Agency (PMRA) of Canada announced the cancellation of the registration of these products in 2018 [10]. It is speculated that the prooxidant activity of OG can disrupt the sensitive structures in microbes, effectively preventing pathogen growth in seeds. Heat sensitization developed in this study will improve the efficacy of antimicrobial practices and achieve safe, rapid, energy-effective, and cost-effective pathogen elimination on seeds or during agriculture or food processing.