A strain previously reported as demonstrating strong

A strain previously reported as demonstrating strong antifungal activity and suitability for food applications is Lactobacillus reuteri R29 (Axel et al., 2016, Oliveira et al., 2015). Oliveira et al. (2015) demonstrated that the cell-free supernatant (cfs) of medium fermented for 48 h with this strain showed antifungal activity against F. culmorum, with PLA being the predominant antimicrobial janex produced. As it belongs to the species L. reuteri, the strain is likely to be a reuterin producer. To date there have been no studies carried out investigating the ability of L. reuteri R29 to produce and accumulate reuterin. Thus, the enhancement of the production and accumulation of antifungal compounds, namely reuterin and PLA, in the bacterial cfs represents a promising opportunity in particular, if this antifungal activity is applicable in food systems. Therefore, the aim of this study was to increase the in vitro antifungal activity of L. reuteri R29 cfs. Different variations of the fermentation medium composition were investigated to increase the accumulation of PLA and reuterin. F. culmorum was used as indicator mould to determine the antifungal activity in vitro. Secondly, characterisations regarding the heat stability of the most efficient cfs were evaluated. Finally, antifungal cfs delivering the best results were applied in the bread system to obtain information regarding suitability for application in a food matrix. This study provides important information regarding the potential of L. reuteri R29 cfs as antifungal agent and its application as a bio-preservative agent, in the bread system.
Materials and methods
Results and discussion
Conclusions In conclusion, this study demonstrates three possibilities for improving the efficiency of antifungal LAB in vitro, using Lactobacillus reuteri R29 as an example. Further understanding regarding production and stability of antifungal compounds was obtained. In particular, the key role of PLA for the antifungal performance of L. reuteri R29 became evident. However, from the MIC90 values of synthetic PLA it also became evident that microbial PLA just in synergy with other bacterial metabolites can serve as efficient antifungal agent. The results achieved in vitro, could only partly be transferred into the bread making process. Reuterin, due to its high reactivity, in particular at high temperatures (Vollenweider et al., 2010), did not lead to satisfactory results in situ. In contrast, the supplementation with Phe, to increase the production of PLA, was found to be very efficient in both in vitro and in situ. Hence, Phe supplemented fermentation media should be considered as promising options to improve the antimicrobial performance of LAB during production of food, such as bread or beverages. The proteolytic activity reported for L. reuteri R29 (Axel et al., 2016) also proposes the possibility to achieve this antifungal effect upon supplementation with Phe rich proteins. This work shows the potential for further exploitation of LAB as bio-preservatives, particularly in environments unsuitable for bacterial fermentation, for example, during grain storage. This demonstrates the potential to enlarge the field of application for the antimicrobial properties of LAB. Further research is required on the in situ production of PLA, including its stability and influence on sensory parameters. In addition, application of such methodologies investigated in this study in further food systems will serve to increase our knowledge in this increasingly pertinent area.
Acknowledgements Financial support for this research was awarded by the Irish Government under the National Development Plan 2007–2013 through the research program FIRM/RSF/CoFoRD. This research was also partly funded by the Irish Department of Agriculture, Food and the Marine.
Introduction Plants have developed an ancient and complex defense strategy through their immune system to combat pathogens and abiotic stresses (de Beer and Vivier, 2011, Lacerda et al., 2014). Among their many defense systems, the production of cationic antimicrobial peptides (cAMPs) is a major contributor to plant resistance to phytopathogens, thanks to their broad spectrum of activity (Stotz et al., 2009). Plant AMPs have been divided into several categories based on their amino acid structure, sequence identity or tertiary structure (Nawrot et al., 2014). Among these AMPs, plant defensins were first discovered in the seeds of wheat and barley (Colilla et al., 1990, Mendez et al., 1990). Plant defensins can be divided into three groups: defensins leading to morphogenic changes in the fungal hyphae, defensins causing reduction of hyphae without morphogenic changes, and defensins without antifungal activity (Broekaert et al., 1995). The expression of plant defensin genes has been reported to be increased in response to pathogens, which supports the idea that these peptides constitute a major defence mechanism (Garcia-Olmedo et al., 1998). In addition, the localization of the plant defensins in different plant organs and tissues, with a preferential cell-wall location in epidermal cells (Lacerda et al., 2014), is highly consistent with a defensive role. The defensins also play a role in the protection against insects, abiotic stress and metal tolerance (Carvalho and Gomes, 2009).