Efficacy of the Combined Protective Cultures of Penicillium chrysogenum and Debaryomyces hansenii for the Control of Ochratoxin A Hazards in Dry-Cured Ham.

The ecological conditions during the ripening of dry-cured ham favour the development of moulds on its surface, being frequently the presence of Penicillium nordicum, a producer of ochratoxin A (OTA). Biocontrol using moulds and yeasts usually found in dry-cured ham is a promising strategy to minimize this hazard. The aim of this work is to evaluate the effect of previously selected Debaryomyces hansenii and Penicillium chrysogenum strains on growth, OTA production, and relative expression of genes involved in the OTA biosynthesis by P. nordicum. P. nordicum was inoculated against the protective cultures individually and combined on dry-cured ham for 21 days at 20 °C. None of the treatments reduced the growth of P. nordicum, but all of them decreased OTA concentration. The lower production of OTA could be related to significant repression of the relative expression of otapksPN and otanpsPN genes of P. nordicum. The efficacy of the combined protective cultures was tested in 24 dry-cured hams in industrial ripening (an 8 month-long production). OTA was detected in nine of the 12 dry-cured hams in the batch inoculated only with P. nordicum. However, in the batch inoculated with both P. nordicum and the combined protective culture, a considerable reduction of OTA contamination was observed. In conclusion, although the efficacy of individual use P. chrysogenum is great, the combination with D. hansenii enhances its antifungal activity and could be proposed as a mixed protective culture to control the hazard of the presence of OTA in dry-cured ham.

Screening of lactic acid bacteria and yeast strains to select adapted anti-fungal co-cultures for cocoa bean fermentation.

Contamination with filamentous fungi during cocoa bean fermentation and drying reduces the quality of cocoa beans and poses a health risk for consumers due to the potential accumulation of mycotoxins. The aim of this study was to develop anti-fungal lactic acid bacteria (LAB)-yeast co-cultures by selecting anti-fungal strains best adapted to the cocoa bean fermentation process from 362 LAB and 384 yeast strains isolated from cocoa bean post-harvest processes. The applied multiphasic screening approach included anti-fungal activity tests in vitro and in vivo and assessment of the carbon metabolism and stress tolerance of the anti-fungal strains in a cocoa pulp simulation medium. The anti-fungal strains, Lactobacillus fermentum M017, Lb. fermentum 223, Hanseniaspora opuntiae H17, and Saccharomyces cerevisiae H290, were selected based on their high fungal growth inhibition capacity and their well-adapted metabolism. Up to seven filamentous fungal strains of the genera Aspergillus, Penicillium, and Gibberella were inhibited on average by 63 and 75% of the maximal inhibition zone by M017 and 223, respectively, and by 25 and 31% by the strains H17 and H290, respectively. Both Lb. fermentum strains converted the medium's glucose, fructose, and citric acid into 20.4-23.0 g/l of mannitol, 3.9-6.2 g/l acetic acid, and 8.6-10.3 g/l lactic acid, whereas the two yeast strains metabolized glucose and fructose to produce 7.4-18.4 g/l of ethanol. The Lb. fermentum strains were further characterized as particularly tolerant towards ethanol, acetic acid, and heat stress and both yeast strains tolerated high amounts of ethanol and lactic acid in the medium. Finally, the anti-fungal in vivo assays revealed that the two Lb. fermentum strains completely inhibited growth of the citrinin-producing strain, P. citrinum S005, and the potentially fumonisin-producing strain, G. moniliformis S003, on the surface of cocoa beans. Furthermore, growth of the aflatoxin-producer A. flavus S075 was inhibited after 10-14 days by all four selected anti-fungal strains, i.e. Lb. fermentum M017, Lb. fermentum 223, H. opuntiae H17, and Sacc. cerevisiae H290, at 51-95% when applied as single cultures and at 100% when the strains were combined into four co-cultures, each composed of a Lb. fermentum and one of the two yeast strains. As a conclusion, these four LAB-yeast co-cultures are recommended for future applications to limit the growth of filamentous fungi and the concomitant mycotoxin production during the fermentation of cocoa beans.

Optimisation of the antifungal potency of the amidated peptide H-Orn-Orn-Trp-Trp-NH2 against food contaminants.

The design of novel efficient antimicrobial peptides (AMPs) faces several issues, such as cost of synthesis, proteolytic stability or cytotoxicity. The identification of key determinants involved in the activity of AMPs, such as cationicity and amphipathicity, allowed the synthesis of short peptides with optimized properties. An ultrashort peptide made of the sequence H-Orn-Orn-Trp-Trp-NH2 (O3TR) showed antifungal activity against several contaminants from food products. This peptide inhibited the growth of the filamentous fungi Fusarium culmorum, Penicillium expansum and Aspergillus niger within a range of concentration of 12.5-50µg/ml. In addition, O3TR inhibited the growth of the yeast Saccharomyces cerevisiae, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii and Kluyveromyces lactis within the range 12.5-50µg/ml. A derivative peptide, called C12O3TR, made by the addition of lauric acid at the N-terminus of O3TR was 2- to 8-fold more active than O3TR against every species. In addition to the inhibition of conidial germination, O3TR and C12O3TR killed F. culmorum hyphae at 100 and 50µg/ml respectively. The MIC of the two peptides against F. culmorum and Z. bailii after heat treatment at 100°C for 60 min and within the pH range 3-10, were not changed. However, the activity of O3TR against F.culmorum and Z. bailii was strongly reduced in salt solutions, whereas the lauric acid peptide kept its antifungal activity and resistance to proteolytic digestion. The conjugation with lauric acid reduced the random coiled structure and increased the α-helical content of O3TR. After conjugation with the dye tetramethylrhodamine (TMR), both peptides entered F. culmorum spores. They also both induced permeabilization of F. culmorum hyphae but only C12O3TR permeabilized Z. bailii membrane. In contrast to the lipopeptide, O3TR did not show haemolytic or cytotoxic activity when applied at the concentrations that exhibited antifungal potency. The two peptides were challenged against a yeast cocktail of S. cerevisiae and Z. bailii, and A. niger in different commercial beverages. After 7 days, O3TR was able to inhibit the yeast cocktail in a commercial lager and carbonated drink. Due to its antifungal potency, high stability and low cytotoxicity, the tetrapeptide could represent a promising starting point of a novel food preservative.

Influence of soiling on boar taint in boars.

It has been suggested that skatole, one of the main compounds responsible for boar taint, can be lowered by keeping pigs clean, as skatole can be absorbed through skin and/or lungs (Hansen, Larsen, Jensen, HansenMoller & Bartongade, 1994). With this experiment, we further investigated this hypothesis by comparing extremely clean with extremely dirty animals with regard to the occurrence of boar taint. One group of boars was washed daily and pens were mucked on and littered down daily (CLEAN), a second group of boars was rubbed with faeces daily (DIRTY) and a third group of boars was kept in control conditions (CONTROL). The treatment was performed during the last four weeks before slaughter. According to the standardised consumer panel evaluations, boars subjected to extra soiling had a higher concentration of boar taint than boars that were kept extra clean. In contrast, expert panels judged general meat flavour to be inferior in CLEAN than CONTROL pigs. The home consumer panel, the hot iron method, and laboratory analyses, i.e., the presence of indole, skatole and androstenone in fat and serum, all showed no significant differences. So no clear indications towards skatole reduction by improving cleanliness of pigs were found.

Cereulide formation by Bacillus weihenstephanensis and mesophilic emetic Bacillus cereus at temperature abuse depends on pre-incubation conditions.

Emetic toxin (cereulide) formation was recently identified in a psychrotolerant species, Bacillus weihenstephanensis [Thorsen, L., Hansen, B.M., Nielsen, K.F., Hendriksen, N.B., Phipps, R.K., Budde, B.B., 2006. Characterization of emetic Bacillus weihenstephanensisis, a new cereulide-producing bacterium. Applied and Environmental Microbiology, 72, 5118-5121.]. Although recent findings indicated B. weihenstephanensis as a cereulide producer only limited information is available regarding environmental conditions affecting cereulide production. In the present study a model agar system was used to compare cereulide production during surface growth of B. weihenstephanensis MC67, and two well known mesophilic cereulide producing Bacillus cereus strains, NC7401 and NS117. Cereulide production was quantified by use of Liquid-Chromatography Mass Spectrometry/Mass Spectrometry. Cereulide production of B. weihenstephanensis MC67 occurred in stationary growth phase, as previously observed for B. cereus, and biomass formation and cereulide formation showed a linear correlation. During incubation at 5 degrees C for 1, 2 and 3 weeks growth was inhibited and as a consequence no detectable cereulide production occurred for any of the three strains. Similar results were obtained for the mesophilic B. cereus strains when incubated at 8 degrees C, whereas B. weihenstephanensis MC67 grew to stationary phase and produced 0.002 microg cereulide/cm(2) agar surface in 1 week. Raising the temperature from 5 degrees C to 25 degrees C for 24 h after 1 week of incubation resulted in growth to stationary phase and production of variable levels of cereulide. B. weihenstephanensis MC67 produced 6.18 microg cereulide/cm(2), B. cereus NS117 0.91 microg cereulide/cm(2) and B. cereus NC7401 0.09 microg cereulide/cm(2). Similar levels of cereulide was produced by the mesophilic strains when raising the temperature from 8 degrees C (instead of from 5 degrees C) to 25 degrees C for 24 h, while a considerably lower level was produced by B. weihenstephanensis MC67 (0.10 microg cereulide /cm(2)). If the temperature was raised from 5 degrees C and 8 degrees C to 25 degrees C for 24 h after an increased incubation time for 2 and 3 weeks, all three strains produced considerably less cereulide. B. weihenstephanensis MC67 produced 100-6000 times less and the mesophilic B. cereus strains produced 9-40 times less cereulide. These results can partly be explained by differences in the growth at the temperature abuse. Effect of chill storage on cereulide production at temperature abuse has not been investigated previously. Results of the present study indicate that storage at 5 and 8 degrees C will not lead to emetic intoxications, however the time at, and choice of chill temperature will determine the amount of cereulide produced in a temperature abuse situation. These results are of relevance for the safety of chilled foods of extended durability.

The co-action of osmotic and high temperature stresses results in a growth improvement of Debaryomyces hansenii cells.

Debaryomyces hansenii is a salt tolerant yeast species, often isolated from sea water or found among other spoilage yeasts in several types of food. In this work, we examined the influence of temperature and increased osmotic pressure (two parameters also important in food industry) on D. hansenii growth. Several other authors showed that its growth at the normal yeast cultivation temperature (28 to 30 degrees C) is stimulated by the presence of sodium, in contrast to the growth of Saccharomyces cerevisiae, which is inhibited by the presence of sodium under the same experimental conditions. Here we show that the previously reported growth stimulation by sodium is temperature dependent in D. hansenii and can be observed under conditions that already amount to high temperature stress for D. hansenii. At a lower temperature (more convenient for D. hansenii cultivation), we found no significant improvement or even an inhibition of cell growth in the presence of Na(+). The growth of D. hansenii at high temperatures is also improved by the presence of potassium or sorbitol. Moreover, the temperature dependence of stimulatory effects of increased osmotic pressure in media does not seem to be unique for D. hansenii; similar relationships between the growth, cultivation temperature and presence of osmolytes we also observed for S. cerevisiae and Schizosaccharomyces pombe.