“Green preservatives” – combating fungi in the food industry by applying antifungal lactic acid bacteria

Fungal spoilage is the most common type of microbial spoilage in food leading to significant economical and health problems throughout the world. Fermentation by lactic acid bacteria (LAB) is one of the oldest and most economical methods of producing and preserving food. Thus, LAB can be seen as an interesting tool in the development of novel bio-preservatives for food industry. The overall objective of this study was to demonstrate, that LAB can be used as a natural way to improve the shelf-life and safety of a wide range of food products. In the first part of the thesis, 116 LAB isolates were screened for their antifungal activity against four Aspergillus and Penicillium spp. commonly found in food. Approximately 83% of them showed antifungal activity, but only 1% showed a broad range antifungal activity against all tested fungi. The second approach was to apply LAB antifungal strains in production of food products with extended shelf-life. L. reuteri R29 strain was identified as having strong antifungal activity in vitro, as well as in sourdough bread against Aspergillus niger, Fusarium culmorum and Penicillium expansum. The ability of the strain to produce bread of good quality was also determined using standard baking tests. Another strain, L. amylovorus DSM19280, was also identified as having strong antifungal activity in vitro and in vivo. The strain was used as an adjunct culture in a Cheddar cheese model system and demonstrated the inhibition of P. expansum. Significantly, its presence had no detectable negative impact on cheese quality as determined by analysis of moisture, salt, pH, and primary and secondary proteolysis. L. brevis PS1 a further strain identified during the screening as very antifungal, showed activity in vitro against common Fusarium spp. and was used in the production of a novel functional wortbased alcohol-free beverage. Challenge tests performed with F. culmorum confirmed the effectiveness of the antifungal strain in vivo. The shelf-life of the beverage was extended significantly when compared to not inoculated wort sample. A range of antifungal compounds were identified for the 4 LAB strains, namely L. reuteri ee1p, L. reuteri R29, L. brevis PS1 and L. amylovorous DSM20531. The identification of the compounds was based on liquid chromatography interfaced to the mass spectrometer and PDA detector

Food preservation using antifungal lactic acid bacteria

Fungal spoilage of food and feed prevails as a major problem for the food industry. The use antifungal-producing lactic acid bacteria (LAB) may represent a safer, natural alternative to the use of chemical preservatives in foods. A large scale screen was undertaken to identify a variety of LAB with antifungal properties from plant, animal and human sources. A total of 6,720 LAB colonies were isolated and screened for antifungal activity against the indicator Penicillium expansum. 94 broad-spectrum producers were identified through 16S rRNA sequencing with the majority of the population comprising Lactobacillus plantarum isolates. Six broad-spectrum isolates were consequently characterised. Pedicococcus pentosaceous 54 displayed potent anti-mould capabilities in pear, plum and grape models and may represent an ideal candidate for use in the beverage industry. Two antifungal Lb. plantarum isolates were assessed for their technological robustness and potential as biopreservatives in refrigerated foods. Lb. plantarum 16 and 62 displayed high levels of tolerance to freeze-drying, low temperature exposure and high salt concentrations. Both lactobacilli were introduced as supplements into orange juice to retard the growth of the spoilage yeast Rhodotorula mucilaginosa. Furthermore the isolates were applied as adjuncts in yoghurt production to successfully reduce yeast growth. Lb. plantarum 16 proved to be the optimal inhibitor of yeast growth in both food matrices. To date there is limited information available describing the mechanisms behind fungal inhibition by LAB. The effects of concentrated cell-free supernatant (cCFS), derived from Lb. plantarum 16, on the growth of two food-associated moulds was assessed microscopically. cCFS completely inhibited spore, germ tube and hyphal development. A transcriptomic approach was undertaken to determine the impact of antifungal activity on Aspergillus fumigatus Af293. A variety of genes, most notably those involved in cellular metabolism, were found to have their transcription modulated in response to cCFS which is indicative of global cellular shutdown. This study provides the first insights into the molecular targets of antifungal compounds produced by LAB. The genome sequence of the steep water isolate Lb. plantarum 16 was determined. The complete genome of Lb. plantarum16 consists of a single circular chromosome of 3,044,738 base pairs with an average G+C content of 44.74 % in addition to eight plasmids. The genome represents the smallest of this species to date while harbouring the largest plasmid complement. Some features of particular interest include the presence of two prophages, an interrupted plantaricin cluster and a chromosomal and plasmid encoded polysaccharide cluster. The sequence presented here provides a suitable platform for future studies elucidating the mechanisms governing antifungal production.

Isolation and characterisation of antifungal compounds from lactic acid bacteria and their application in wheat and gluten-free bread

As part of the “free-from” trend, biopreservation for bread products has increasingly become important to prevent spoilage since artificial preservatives are more and more rejected by consumers. A literature review conducted as part of this thesis revealed that the evaluation of more suitable antifungal strains of lactic acid bacteria (LAB) is important. Moreover, increasing the knowledge about the origin of the antifungal effect is fundamental for further enhancement of biopreservation. This thesis addresses the investigation of Lactobacillus amylovorus DSM19280, Lb. brevis R2: and Lb. reuteri R29 for biopreservation using in vitro trials and in situ sourdough fermentations of quinoa, rice and wheat flours as biopreservatives in breads. Their contribution to quality and shelf life extension on bread was compared and related to their metabolic activity and substrate features. Moreover, the quantity of antifungal carboxylic acids produced during sourdough fermentation was analysed. Overall a specific profile of antifungal compounds was found in the sourdough samples which were strain and substrate dependently different. The best preservative effect in quinoa sourdough and wheat sourdough bread was achieved when Lb. amylovorus DSM19280 fermented sourdough was used. However, the concentration of the antifungal compounds found in these biopreservatives were much lower when compared with Lb. reuteri R29 as the highest producer. Nevertheless, the artificial application of the highest concentration of these antifungal compounds in chemically acidified wheat sourdough bread succeeded in a longer shelf life than achieved only by acidifying the dough. This evidences their partial contribution to the antifungal activity and their synergy. Additionally, a HRGC/MS method for the identification and quantification of the antifungal active compounds cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) was successfully developed by using stable isotope dilutions assays with the deuterated counterparts. It was observed that the concentrations of cyclo(Leu-Pro), cyclo(Pro-Pro), and cyclo(Phe-Pro) increased only moderately in MRS-broth and wort fermentation by the activity of the selected microorganism, whereas the concentration of cyclo(Met-Pro) stayed unchanged.

Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity.

Immune responses must be well restrained in a steady state to avoid excessive inflammation. However, such restraints are quickly removed to exert antimicrobial responses. Here we report a role of autophagy in an early host antifungal response by enhancing NFκB activity through A20 sequestration. Enhancement of NFκB activation is achieved by autophagic depletion of A20, an NFκB inhibitor, in F4/80(hi) macrophages in the spleen, peritoneum and kidney. We show that p62, an autophagic adaptor protein, captures A20 to sequester it in the autophagosome. This allows the macrophages to release chemokines to recruit neutrophils. Indeed, mice lacking autophagy in myeloid cells show higher susceptibility to Candida albicans infection due to impairment in neutrophil recruitment. Thus, at least in the specific aforementioned tissues, autophagy appears to break A20-dependent suppression in F4/80(hi) macrophages, which express abundant A20 and contribute to the initiation of efficient innate immune responses.

Clinical effectiveness of posaconazole versus fluconazole as antifungal prophylaxis in hematology-oncology patients: a retrospective cohort study.

In preventing invasive fungal disease (IFD) in patients with acute myelogenous leukemia (AML) or myelodysplastic syndrome (MDS), clinical trials demonstrated efficacy of posaconazole over fluconazole and itraconazole. However, effectiveness of posaconazole has not been investigated in the United States in real-world setting outside the environment of controlled clinical trial. We performed a single-center, retrospective cohort study of 130 evaluable patients ≥18 years of age admitted to Duke University Hospital between 2004 and 2010 who received either posaconazole or fluconazole as prophylaxis during first induction or first reinduction chemotherapy for AML or MDS. The primary endpoint was possible, probable, or definite breakthrough IFD. Baseline characteristics were well balanced between groups, except that posaconazole recipients received reinduction chemotherapy and cytarabine more frequently. IFD occurred in 17/65 (27.0%) in the fluconazole group and in 6/65 (9.2%) in the posaconazole group (P = 0.012). Definite/probable IFDs occurred in 7 (10.8%) and 0 patients (0%), respectively (P = 0.0013). In multivariate analysis, fluconazole prophylaxis and duration of neutropenia were predictors of IFD. Mortality was similar between groups. This study demonstrates superior effectiveness of posaconazole over fluconazole as prophylaxis of IFD in AML and MDS patients. Such superiority did not translate to reductions in 100-day all-cause mortality.

A polysaccharide extracted from sphagnum moss as antifungal agent in archaeological conservation

On the basis of the well-known preservative properties of Sphagnum moss, a potential opportunity to use moss polysaccharides (Sphagnan) in art conservation was tested. Polysaccharides were extracted from the moss (S. palustre spp.) in the amount of 4.1% of the Sphagnum plant dry weight. All lignocelluloses were removed from this extract as a result of the treatment of the moss cellulose with sodium chlorite. The extracted polysaccharide possessed a strong acidic reaction (pH 2.8) and was soluble in water and organic solvents. The extract was tested on laboratory bacterial cultures by the disk-diffusion method. The antibacterial effect was demonstrated for E. coli and P. aeruginosa (both gram-negative) while Staphylococcus aurelus (gram-positive) was shown to be insensitive to Sphagnum polysaccharides. The antifungal effect of Sphagnum extract was tested by the disk-diffusion method on the spores of seventeen fungal species. These fungi were isolated from ethnographic museum objects and from archaeological objects excavated in the Arctic. Twelve of these isolates appeared susceptible to the extract. The inhibiting effect of the extract was also tested by the modified broth-dilution method on the most typical isolate (Aspergillus spp.). In this experiment, in one ml of the nutritious broth, 40µl of 3% solution of polysaccharides in water killed 10,000 fungal spores in 6 hours. The inhibiting effect was not connected to the acidity or osmotic effect of Sphagnum polysaccharides. As an example of the application of Sphagnum polysaccharides in art conservation, they were added as preservative agents to conservation waxes. After three weeks of exposure of microcrystalline wax to test fungi (Aspergillus spp.), 44% of wax was consumed. When, however, ~ 0.1% (w/w) of Sphagnum extract was mixed with wax, the weight loss of wax was only 4% in the same time interval. On the basis of this study it was concluded that Sphagnum moss and Sphagnum products can be recommended for use in art conservation as antifungal agents.

First report of a novel plant lysozyme with both antifungal and antibacterial activities.

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4 kDa in SDS–polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355 U/mg at pH 5.5 and 30 °C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 °C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.

Antifungal photodynamic therapy

In photodynamic antimicrobial chemotherapy (PACT), a combination of a sensitising drug and visible light causes selective destruction of microbial cells. The ability of light-drug combinations to kilt microorganisms has been known for over 100 years. However, it is only recently with the beginning of the search for alternative treatments for antibiotic-resistant pathogens that the phenomenon has been investigated in detail. Numerous studies have shown PACT to be highly effective in the in vitro destruction of viruses and protozoa, as well as Gram-positive and Gram-negative bacteria and fungi. Results of experimental investigations have demonstrated conclusively that both dermatomycetes and yeasts can be effectively killed by photodynamic action employing phenothiazinium, porphyrin and phthatocyanine photosensitisers. Importantly, considerable setectivity for fungi over human cells has been demonstrated, no reports of fungal resistance exist and the treatment is not associated with genotoxic or mutagenic effects to fungi or human cells. In spite of the success of cell culture investigations, only a very small number of in vivo animal. and human trials have been published. The present paper reviews the studies published to date on antifungal applications of PACT and aims to raise awareness of this area of research, which has the potential to make a significant impact in future treatment of fungal infections. (c) 2007 Elsevier GmbH. All rights reserved.

Anti-adherent and antifungal activities of surfactant-coated poly (ethylcyanoacrylate) nanoparticles

Application of non-drug-loaded poly(ethylcyanoacrylate) nanoparticles (NP) to buccal epithelial cells (BEC) imparted both anti-adherent and antifungal effects. NP prepared using emulsion polymerisation and stabilised using cationic, anionic and non-ionic surfactants decreased Candida albicans blastospore adhesion, an effect attributable to the peripheral coating of surfactant. Cetrimide and Pluronic (R) P 123 were shown to be most effective, producing mean percentage reductions in blastospore adherence of 52.7 and 37.0, respectively. Resultant zeta potential matched the polarity of the surfactant, with those stabilised using cetrimide being especially positive (+31.3 mV). Preparation using anionic surfactants was shown to be problematic, with low yield and wide particle size distribution. Evaluation of the antifungal effect of the peripheral coat was evaluated using zones of inhibition and viable counts assays. The former test revealed poor surfactant diffusion through agar, but did show evidence of limited kill. However, the latter method showed that cationic surfactants associated with NP produced high levels of kill, in contrast to those coated with anionic surfactants, where kill was not evident. Non-ionic surfactant-coated NP produced intermediate kill rates. Results demonstrate that surfactant-coated NP, particularly the cationic types, form the possible basis of a prophylactic formulation that primes the candidal target (BEC) against fungal adhesion and infection. (c) 2007 Elsevier B.V. All rights reserved.

The impact of a PCR assay for candidemia on antifungal drug prescribing in critical care: an interrupted time series pilot study.

Objective To evaluate the feasibility of conducting a definitive study to assess the impact of introducing a rapid PCR-based test for candidemia on antifungal drug prescribing. Method Prospective, single centre, interrupted time series study consisting of three periods of six months' duration. The assay was available during the second period, during which the PCR assay was available for routine use by physicians Monday–Friday with guaranteed 24-h turnaround time. For each period total antifungal drug use, expressed as treatment-days, was recorded and an adjustment was made to exclude estimated use for proven candidemia. Also, during the intervention period, antifungal prescribing decisions for up to 72 h after each PCR result became available were recorded as either concordant or discordant with that result. Results While overall antifungal use remained relatively stable throughout, after adjustment for candidemia, there was a 38% reduction in use following introduction of the PCR test; however, this was nonsignificant at the 95% level. During the intervention period overall concordance between the PCR result and prescribing decisions was 84%. Conclusions The PCR assay for candidemia was requested, prescribing decisions were generally concordant with the results produced and there was an apparent decrease in antifungal prescription, although this was sustained even after withdrawal of the intervention; these findings should be more thoroughly evaluated in a larger trial.

Trends in candidemia and antifungal susceptibility in a university hospital in Northern Ireland 2001-2006

Objectives: To describe the species distribution and antifungal susceptibility trends for documented episodes of candidemia at the Royal Hospitals, Belfast, 2001-2006. Methods: Laboratory-based retrospective observational study of all episodes of candidemia. Results: There were 151 episodes of candidemia. The species recovered were: 96 C. albicans; 26 C. glabrata; 18 C. parapsilosis; five C. tropicalis; four C. guilliermondii; one C. famata and one C. dubliniensis. We separated the data into two periods 2001-2003 and 2004-2006; contrary to the findings of other investigators, there was a notable trends toward increasing frequency of C. albicans and decreasing frequency of non-albicans species over time. Although the proportion of C. albicans, C. parapsilosis and C. tropicalis isolates susceptible to fluconazole was unchanged over time, a trend of decreased susceptibility of C. glabrata to fluconazole was noted over the six-year period. Overall, 73% and 7.7% of C. glabrata isolates had susceptible-dose-dependent and resistant phenotypes, respectively. The percentage of C. glabrata isolates susceptible to fluconazole (MIC

Chaotropic and hydrophobic stress mechanisms of antifungal substances

Growth and metabolism of fungi can be curtailed by chaotropic solutes and hydrophobic substances, both of which can weaken or inhibit non-covalent interactions within and between macromolecular systems. Here we explore the potential to utilize the fungistatic and fungicidal activities of such stressors as the basis for commercial formulations. A method was developed for the quantification of chaotropicity, which can be used for chemically diverse substances, in order elucidate roles of chaotropicity and hydrophobicity in microbial ecology (both of which are sufficiently potent to limit the Earth’s microbial biosphere). A large number of naturally occurring substances act as chaotropic or hydrophobic stressors including aliphatic alcohols, salts such as MgCl2, aromatics such as phenol, and hydrocarbons such as hexane and octene. We suggest that these stress parameters provide the (hitherto unidentified) modes-of-action for some extant antifungal products. The findings are discussed in relation to the development of a new generation of antifungals.