Brid Brosnan

Antifungal activity of Lactobacillus against Microsporum canis, Microsporum gypseum and Epidermophyton floccosum

A total of 220 lactic acid bacteria isolates were screened for antifungal activity using Aspergillus fumigatus and Aspergillus niger as the target strains. Four Lactobacillus strains exhibited strong inhibitory activity on agar surfaces. All four were also identified as having strong inhibitory activity against the human pathogenic fungi Microsporum canis, Microsporum gypseum and Epidermophyton floccosum. One of the four lactobacilli, namely Lb. reuteri ee1p exhibited the most inhibition against dermatophytes. Cell-free culture supernatants of Lb. reuteri ee1p and of the non-antifungal Lb. reuteri M13 were freeze-dried and used to access and compare antifungal activity in agar plate assays and microtiter plate assays. Addition of the Lb. reuteri ee1p freeze-dried cell-free supernatant powder into the agar medium at concentrations greater than 2% inhibited all fungal colony growth. Addition of the powder at 5% to liquid cultures caused complete inhibition of fungal growth on the basis of turbidity. Freeze-dried supernatant of the non-antifungal Lb. reuteri M13 at the same concentrations had a much lesser effect. As Lb. reuteri M13 is very similar to the antifungal strain ee1p in terms of growth rate and final pH in liquid culture, and as it has little antifungal activity, it is clear that other antifungal compounds must be specifically produced (or produced at higher levels) by the anti-dermatophyte strain Lb. reuteri ee1p. Reuterin was undetectable in all four antifungal strains. The cell free supernatant of Lb. reuteri ee1p was analyzed by LC-FTMS using an Accela LC coupled to an LTQ Orbitrap XL mass spectrometer. The high mass accuracy spectrum produced by compounds in the Lb. reuteri ee1p strain was compared with both a multianalyte chromatogram and individual spectra of standard anti-fungal compounds, which are known to be produced by lactic acid bacteria. Ten antifungal metabolites were detected.

The QuEChERS approach in a novel application for the identification of antifungal compounds produced by lactic acid bacteria cultures

Lactic Acid Bacteria (LAB) play an important role as natural food preservatives in many fermented food systems. To-date, characterisation of their diverse range of metabolites has been limited. Improved quantitation of low, medium and high concentration antifungal compounds is required, ensuring that both known and unknowns compounds are identified. This manuscript reports the first application of QuEChERS (quick, easy, cheap, effective, rugged and safe) for the extraction of natural antifungal metabolites in LAB cultures. The method provides improved individual recoveries (>78%) for 15 known antifungal compounds, an improvement of 26% compared to previously reported techniques (>52%). A protocol was developed that allowed LAB cultures to be easily assessed on a fully validated high performance liquid chromatography with ultra violet/diode array detection (HPLC-UV/DAD) method. Previously reported methods involving direct injection of filtered extracts and SPE clean-up, suffered from a rise in chromatographic baseline due to interfering matrix components, limiting accurate quantitation. This QuEChERS method removed these interfering matrix components to deliver clean chromatograms with greater recoveries (78.2-127.4%) and lower RSD values (2.5-10.8%) of all 15 antifungal compounds. The validated method was applied to LAB strains showing particularly strong antifungal activity and provided an increase in the number of compounds detected (both known and unknown) compared to previous techniques for the same strains, due to the improved recoveries now possible by this method. Confirmation of the compounds identified was performed by analysis on a liquid chromatography linear ion trap quadrupole Orbitrap hybrid Fourier transform mass spectrometer (LC-FTMS). This first application of QuEChERS to LAB cultures has significantly improved the analytical capabilities of antifungal compound profiling especially where the synergy of numerous compounds is suspected as producing the observed activity. LAB cultures can now be easily integrated into various food matrices, as natural food preservatives, now that a complete analyte profile is achievable.

Antifungal sourdough lactic acid bacteria as biopreservation tool in quinoa and rice bread.

The use of sourdough fermented with specific strains of antifungal lactic acid bacteria can reduce chemical preservatives in bakery products. The main objective of this study was to investigate the production of antifungal carboxylic acids after sourdough fermentation of quinoa and rice flour using the antifungal strains Lactobacillus reuteri R29 and Lactobacillus brevis R2Δ as bioprotective cultures and the non-antifungal L. brevis L1105 as a negative control strain. The impact of the fermentation substrate was evaluated in terms of metabolic activity, acidification pattern and quantity of antifungal carboxylic acids. These in situ produced compounds (n=20) were extracted from the sourdough using a QuEChERS method and detected by a new UHPLC-MS/MS chromatography. Furthermore, the sourdough was applied in situ using durability tests against environmental moulds to investigate the biopreservative potential to prolong the shelf life of bread. Organic acid production and TTA values were lowest in rice sourdough. The sourdough fermentation of the different flour substrates generated a complex and significantly different profile of carboxylic acids. Extracted quinoa sourdough detected the greatest number of carboxylic acids (n=11) at a much higher concentration than what was detected from rice sourdough (n=9). Comparing the lactic acid bacteria strains, L. reuteri R29 fermented sourdoughs contained generally higher concentrations of acetic and lactic acid but also the carboxylic acids. Among them, 3-phenyllactic acid and 2-hydroxyisocaproic acid were present at a significant concentration. This was correlated with the superior protein content of quinoa flour and its high protease activity. With the addition of L. reuteri R29 inoculated sourdough, the shelf life was extended by 2 days for quinoa (+100%) and rice bread (+67%) when compared to the non-acidified controls. The L. brevis R2Δ fermented sourdough bread reached a shelf life of 4 days for quinoa (+100%) and rice (+33%). However, the shelf life was similar to the chemically acidified control indicating that the preservation effect of the carboxylic acids seems to have a minor contribution effect on the antifungal activity in gluten-free breads.

High-resolution mass spectrometry analysis of tetrodotoxin (TTX) and its analogues in puffer fish and shellfish

ABSTRACT Tetrodotoxin (TTX) is an emerging toxin in the European marine environment. It has various known structural analogues. It acts as a sodium channel blocker; the ability of each analogue to bind to the sodium channel varies with the particular structure of each analogue. Thus, each analogue will vary in its toxic potential. TTX analogues co-occur in food samples at variable concentrations. An LC-MS method was developed for the identification and quantitation of several analogues of TTX using an LTQ-Orbitrap XL mass spectrometer. The LTQ-Orbitrap XL mass spectrometer facilitates high mass accuracy measurement up to 100,000 full width at half maximum (FWHM). Using high resolution at 100,000 FWHM allows for the identification of TTX and its analogues in various matrices, including puffer fish and molluscan shellfish samples (Δ ppm = 0.28–3.38). The confirmation of characteristic fragment ions of TTX and its analogues was achieved by determining their elemental formulae via high mass accuracy. A quantitative method was then developed and optimised using these characteristic fragment ions. The limit of quantitation (LOQ) of the method was 0.136 µg g–1 (S/N = 10) and the limit of detection (LOD) was 0.041 µg g–1 (S/N = 3) spiking TTX standard into TTX-free mackerel fish extracts. The method was applied to naturally contaminated puffer fish and molluscan shellfish samples to confirm the presence of TTX and its analogues.

LC-MS/MS method for the determination of tetrodotoxin (TTX) on a triple quadruple mass spectrometer

ABSTRACT Tetrodotoxin (TTX), often referred to as the ‘puffer fish’ poison, is a marine toxin and it has been identified as the agent responsible for many food poisoning incidents around the world. It is a neurotoxin that blocks voltage-gated sodium channels, resulting in respiratory paralysis and even death in severe cases. It is known to occur in many different species of fish and other organisms. The toxin is mainly found in the Southeast Asia region. Worryingly, TTX is starting to appear in European waters. It is suspected that this is a consequence of Lessepsian migration, also known as the Erythrean invasion. Therefore, straightforward and reliable extraction and analytical methods are now urgently required to monitor seafood of European origin for TTX. This paper provides a versatile, dependable and robust method for the analysis of TTX in puffer fish and trumpet shellfish using LC-MS/MS. A three-stage approach was implemented involving: (1) the screening of samples using fast multiple reaction monitoring (MRM) mass spectral analysis to identify quickly positive samples on a triple quadrupole mass spectrometer (QqQMS/MS), the API 3000; (2) a Fourier-transform (FT)-MS full-scan analysis of positive samples to collect qualitative data; and (3) a method with a longer chromatography run to identify and quantitate the positive samples using the QqQMS. The quantitative LC-QqQMS method delivered excellent linearity for solvent-based standards (0.01–7.5 µg ml–1; R2 ≥ 0.9968) as well as for matrix-matched standards (0.05–37.50 µg g–1; R2 ≥ 0.9869). Good inter-day repeatability was achieved for all the relevant analytes with %RSD values (n = 9) ranging from 1.11% to 4.97% over a concentration range of 0.01–7.5 µg ml–1. A sample clean-up procedure for the puffer fish and trumpet shellfish was developed to ensure acceptable and reproducible recoveries to enable accurate and precise determination of TTX in a myriad of tissues types. Blank mackerel matrix was used for the TTX standard spiking studies in order to calculate the recoveries of the toxin during the extraction procedure. The recovery was 61.17% ± 5.42% for the extraction protocol. MS/MS studies were performed on a linear-trap quadruple-Orbitrap mass spectrometer (LTQ-Orbitrap) to obtain high-mass-accuracy data of the target analytes and their characteristic fragment ions in the puffer fish and trumpet shellfish samples. This facilitated identification of TTX and its associated analogues. These high-mass-accuracy studies facilitated the development of a rapid MRM-based quantitative method for TTX determination on the LC-QqQMS.

A comprehensive investigation into sample extraction and method validation for the identification of antifungal compounds produced by lactic acid bacteria using HPLC-UV/DAD

High Performance Liquid Chromatography with Ultra Violet/Diode Array Detection (HPLC-UV/DAD) is an analytical tool that has the potential to develop and advance analytical research and routine analyses in industrially-important Lactic Acid Bacteria (LAB) with the aim of detecting metabolites with antifungal capabilities. Diode array detection is a multi-wavelength detection system that enables the simultaneous collection of absorption data from a range of UV and visible (UV/vis) wavelengths. The majority of antifungal organic compounds have characteristic UV/vis absorption spectra due to the presence of chromophoric groups or structures, this feature can be utilised to assist in identifying and quantifying the components (known and unknown) as they pass through the detector. HPLC-UV/DAD is robust, cheap to purchase and run, easy to maintain, the software is user friendly and automated for quantitation and the methodology can be easily switched/up-scaled to semi-preparative applications for target analyte isolation and purification. However inefficient sample preparation has become the limiting factor for the routine use of HPLC-UV/DAD in the LAB area. This manuscript will outline the development and validation of an HPLC-UV/DAD method that addresses all of the factors that has limited the routine use of this technology for LAB samples. The method has been developed to quantitatively profile fifteen antifungal compounds in LAB strains in a run time of 40 minutes, allowing the assessment of crude samples (direct injection), eliminating sample matrix chromatographic baseline rise effects through the optimisation of liquid–liquid extraction (LLE with average recoveries = 87%) and assessing a widely applied SPE methodology that is shown to yield poor recoveries for the suite of target analytes. The method reported is validated (linear calibration curve: 1–50 μg mL−1; R2 ≥ 0.999; intra-day repeatability gave RSD values (n = 3) ranging from 0–8.8%; inter-day repeatability gave RSD values (n = 9) ranging from 0.3–7.9%; LODs of 0.2–0.6 μg mL−1 and LOQs of 0.5–0.9 μg mL−1 following LLE applied to a range of crude culture broths and extracted LAB cultures, and all results were confirmed against a liquid chromatography (LC) linear ion trap quadrupole (LTQ) Orbitrap hybrid Fourier transform mass spectrometer (FTMS). In summary, through this methodology, HPLC-UV/DAD can be routinely used as a quantitative screening technique for both culture broth (e.g. de Man, Rosaga and Sharpe (MRS)) and LLE-extracted LAB cultures.

Improvement of taste and shelf life of yeasted low-salt bread containing functional sourdoughs using Lactobacillus amylovorus DSM 19280 and Weisella cibaria MG1

The challenge remains for the baking industry to reduce salt levels in yeasted bread as directed by governments, retailers and consumers around the world. The two main problems associated with the reduction of salt are a lack of salty taste and the reduction in shelf-life. Both of these issues are addressed in the presented work. A range of breads containing different levels of salt (0.0%, 0.3% and 1.2% of NaCl) in combination with various levels of sourdough (0%, 6%, 12%, 18%, 24%) was produced. The different doughs were analysed for their rheological behaviour. The bread quality characteristics such as loaf volume, crumb structure, staling rate and microbial shelf life were also determined. The sourdoughs were analysed for their different metabolites: organic acids, sugars, exopolysaccharides (EPS), and antifungal compounds. A trained sensory panel was used to perform descriptive analysis of the bread samples. The object of this paper is to use functional sourdoughs, containing Lactobacillus amylovorus DSM 19280 and Weisella cibaria MG1 to compensate for the quality problems that occur when salt is reduced in yeasted bread. The application of functional sourdoughs containing exopolysaccharides and/or antifungal substances in salt reduced breads significantly improved the quality. The application of functional sourdoughs allows the reduction of salt to a level of 0.3%.