Variability of the lipolytic activity in Yarrowia lipolytica and its dependence on environmental conditions

This work was aimed to the evaluation of the variability of lipolytic activity in Yarrowia lipolytica strains, as well as to asses for a selected strain, the response to the changes of physico-chemical variables (such as pH, NaCl and lipid content), in order to obtain predictive models describing their effects on the lipolysis pattern. The strains tested, having different environmental origin, showed different patterns of the free fatty acids (FFA) released. The clustering of the free fatty acids profiles evidenced that the unweighted average distance within the strains of the same species did not exceeded 30%. However, the lipolytic activity of some strains generated FFA profiles that differentiated from the majority of the strains considered. Also, when a single strain was inoculated in model systems in which pH, NaCl and milk fat were modulated according to a Central Composite Design (CCD), chemico-physical characteristics of the system led to marked variations in the lipolytic activity with consequent changes in individual fatty acids released. In most cases, when the same Y. lipolytica strain was used, under the experimental conditions adopted, the modulation of the lactic acid, NaCl and lipid content did not generate differences in the fatty acid release exceeding 20-21%. However, some combinations of factors remarkably affected lipase expression or activity, and generated differences in the fatty acid released higher than those observed among different strains of the same species.     

Predicting fungal growth: the effect of water activity on Penicillium roqueforti.

The effect of water activity on the colony growth of Penicillium roqueforti is studied by predictive modelling techniques. Measured colony diameter growth curves are fitted to estimate the growth rate and lag phase of the curves. The colony growth rate was modelled by a quadratic function of transformed water activity (a(w)) values, as suggested by Baranyi et al. (Food Microbiol. 10 (1993) 43-59). The lag time was modelled as a function of water activity, by means of the sum of a constant term and a hyperboloid function of a(w) raised to the second power. The lag-phase of Penicillium roqueforti was found insensitive to the water activity in the range of its higher (a(w) > 0.92) values.     

Characterization of Penicillium roqueforti strains used as cheese starter cultures by RAPD typing

Seventy-six strains of Penicillium roqueforti used as starter cultures for mould ripened blue cheeses have been analysed for their RAPD genotype by using three different primers. A comparison of the RAPD patterns within each primer group revealed that the genetic constitution of the strains was similar, as most of the strains showed very similar overall patterns. Despite these similarities with each primer, distinct RAPD genotype groups could be identified. With one of the primers, it was possible to detect two heteropolymorphic DNA regions resulting in 13 different groups. With the other two primers, three or four groups could be identified. Between the groups of the different primers marked correspondence with respect to strain distribution could be observed, indicating that the polymorphisms detected by the primers were not independent. The RAPD patterns were compared to the production of secondary metabolites. A correlation was observed between the RAPD patterns of all primers and the production of mycophenolic acid. In addition, one of the primer (ari1) was able to distinguish between P. roqueforti strains producing larger or smaller numbers of metabolites.     

Mycotoxin production capability of Penicillium roqueforti in strains isolated from mould-ripened traditional Turkish civil cheese

Mould-ripened civil is a traditional cheese produced mainly in eastern Turkey. The cheese is produced with a mixture of civil and whey curd cheeses (lor). This mixture is pressed into goat skins or plastic bags and is ripened for more than three months. Naturally occurring moulds grow on the surface and inside of the cheese during ripening. In this research, 140 Penicillium roqueforti strains were isolated from 41 samples of mould-ripened civil cheese collected from Erzurum and around towns in eastern Turkey. All strains were capable of mycotoxin production and were analysed using an HPLC method. It was established that all the strains (albeit at very low levels) produced roquefortine C, penicillic acid, mycophenolic acid and patulin. The amounts of toxins were in the ranges 0.4–47.0, 0.2–43.6, 0.1–23.1 and 0.1–2.3 mg kg^?1, respectively. Patulin levels of the samples were lower than the others. The lowest level and highest total mycotoxin levels were determined as 1.2 and 70.1 mg kg^?1 respectively. The results of this preliminary study may help in the choice of secondary cultures for mould-ripened civil cheese and other mould-ripened cheeses.     

Penicillium camemberti and Penicillium roqueforti enhance the growth and survival of Shiga toxin-producing Escherichia coli O157 under mild acidic conditions

The effects of secondary starter molds of common mold-ripened cheeses on the Shiga toxin-producing Escherichia coli (STEC) O157 were assessed in 3 model systems. In the 1st model, 8 STEC O157 strains were incubated in the spent culture of Penicillium camemberti or Penicillium roqueforti under mild acidic conditions at 25 °C. In the spent cultures of the mold at pH 4.8 to 5.0, the lag times of STEC O157 growth were significantly shorter than those observed in fresh medium. Analyses of the spent culture of P. camemberti showed that the causative agents of the growth enhancement were produced by the mold in response to an acidic environment and were not fully inactivated in heat treatment. In the 2nd model, P. camemberti and STEC O157 were cocultured in acidified milk at 25 °C. The population of STEC O157 reached 10(8) CFU/mL in the presence of the mold, whereas the population steadily declined in the absence of the mold. Although this growth enhancement was partially attributable to alkalization by the mold, it was observed even when the pH of this model was stabilized. In the 3rd model, 2 STEC O157 strains were incubated in the spent cultures of molds at pH 4.5 at 10 °C. In the spent culture, proportions of injured cells were significantly lower and D values were significantly higher than those in control, except one STEC O157 strain in the spent culture of P. camemberti. These results showed that the molds could enhance the growth and survival of STEC O157 by changing the environment. Practical Application: This study demonstrated that molds in foods can improve the growth and survival of the Shiga toxin-producing Escherichia coli O157. Because microbial interactions are ubiquitous in food, our results provide an important insight for understanding the behavior of microorganisms in food.     

Mycotoxin-forming ability of two Penicillium roqueforti strains in blue moldy tulum cheese ripened at various temperatures

Isolated and identified toxigenic and nontoxigenic Penicillium roqueforti (PR) strains from moldy tulum cheeses were inoculated into tulum cheeses made in the laboratory and ripened at 5 and 12 degrees C. Mycotoxin (patulin, penicillic acid, PR toxin, and roquefortine) formation in the control and mold-inoculated cheeses were detected by thin-layer chromatography on the first through fourth months of ripening. Patulin, penicillic acid, and PR toxin were not detected in the experimental cheeses. Only roquefortine was detected in cheese inoculated with the toxigenic strain of the mold and ripened at 5 and 12 degrees C on the third and first months of ripening, respectively. Toxin in cheeses ripened at 5 and 12 degrees C was 2.1 to 2.4 and 2.1 to 3.8 mg/kg cheese, respectively.     

Incidence of Penicillium roqueforti and roquefortine C in silages

Wilted grass and whole-crop maize silages taken from farm silos in northern Germany were analysed for fermentation pattern, mould counts and composition of mycoflora as well as for roquefortine C. In general, increasing DM contents of visibly unmoulded silages resulted in decreasing amounts of volatile fatty acids and a greater portion of samples with a high number of mould propagules. The average mould count of these silages was found to be 1·4×10⁴ cfu g⁻¹, whereas visibly moulded samples contained about 1×10⁸ cfu g⁻¹. Penicillium roqueforti was the predominating fungal species in silages occasionally accompanied by species of the genera Aspergillus, Mucor, Monascus and/or Geotrichum. Penicillium roqueforti was detected in 89% of the visibly moulded and in 85% of the visibly unmoulded samples. Of 24 visibly moulded silages tested, 21 samples contained roquefortine C, a mycotoxin known to be produced by P roqueforti. The highest level of roquefortine C found was 36 mg kg⁻¹ DM. Even 6 of 24 visibly unmoulded samples analysed for this mycotoxin were contaminated with roquefortine C but only in trace amounts. Roquefortine C is considered as a model compound for the biosynthesis of toxic fungal metabolites produced by P roqueforti in silages. The P roqueforti-count can be employed as a criterion to predict the contamination of silages with mycotoxins produced by this fungal species. © 1998 SCI.     

The effects of casein on the metabolism of fatty acids,methyl ketones and secondary alcohols by Penicillium roqueforti in buffered solutions

The metabolism of fatty acids and methyl ketones by Penicillium roqueforti was studied in phosphate buffered solutions and buffered solutions with added soluble casein. It was found that casein enhanced the metabolism of fatty acids and the quantity of methyl ketones produced. Methyl ketones were also found to be metabolised by P. roqueforti in phosphate buffered solutions. The addition of fatty acids inhibited the metabolism of the ketones. The inhibitory effects of the fatty acids were nullified in the presence of casein hydrolysate.     

Variability of the lipolytic activity in Yarrowia lipolytica strains in pork fat

This work studied the variability in lipolytic activity in 35 strains of Yarrowia lipolytica inoculated in pork fat after 7 and 21 days of storage at 15 °C. The strains were able to generate three different hydrolysis profiles. In particular, the strains PO10, PO14, RO1, RO5, Y15, Y16A, Y20, B5, 7B, 7B3, 16B and 21C caused an increase with time in concentrations of C16:0, C18:0, C18:1, C18:1(Δ11) and C18:2 which were the predominant free fatty acids (FFAs). On the contrary, the strains PO1, PO19, PO23, RO22, Y12, B4, B74, GB, 5B, 5D, 27D and W29 showed an opposite trend, while the remaining ones induced no change. Because the released FFAs can be considered precursors for flavour development, the results suggest the potential use of some Y. lipolytica strains in sausage making to improve the overall aroma.     

Enzymes of Penicillium roqueforti involved in the biosynthesis of cheese flavor.

The ripening of blue and Roquefort cheeses is accomplished by the concerted and controlled actions of enzymes of the mold Penicillium roqueforti. The properties and effects of the enzymes involved in flavor development (i.e., proteases, lipase and beta-ketoacid decarboxylase) are reviewed. The metabolic activities of both spores and mycelia of P. roqueforti in relation to fatty acid metabolism and flavor generation are discussed. The chemical composition of blue cheese flavor and the simulation of this flavor by fermentation and formulation are briefly surveyed. Some nutritional aspects of blue cheese are cited.     

Changes in biochemical components during the germination of spores of Penicillium roqueforti

Germination of spores of Penicillium roqueforti consisted of three stages, i.e. swelling, germ-tube emergence and hyphal elongation which occurred at 4, 12 and 16 h respectively after initiation of incubation of resting spores in the germination medium. The swelling phase represents the period during which the germinating spore assembled required synthetic enzymes though there was only a small increase in dry weight (2 mg h^?1 per 10⁹ spores). Nuclear division occurred since DNA multiplied rapidly during spore swelling. The percentages of RNA and proteins reached their maxima at the end of this period. The unsaturated fatty acids, e.g. linoleic and linolenic acids, were synthesised. Germ-tube emergence represented a transition phase during which growth was moderate (at a rate of 9 mg h^?1 per 10⁹ spores) as compared to hyphal elongation phase (34 mg h^?1 per 10⁹ spores). The percentage of RNA, proteins and carbohydrates decreased. The percentage of unsaturated fatty acids was highest at the middle of this period and then declined. The hyphal elongation phase represented a period of rapid growth. The relative contents of proteins and DNA remained constant while the percentage of carbohydrates and saturated fatty acids increased, reflecting the proliferation of cellular and membrane materials.     

Variability of the lipolytic activity and volatile molecules production by a strain of Yarrowia lipolytica in pork fat and its dependence on environmental conditions

The effects of selected variables, i.e. temperature, water activity and yeast inoculation level, on the lipolytic pattern and volatile production by Yarrowia lipolytica Y16A (chosen on the basis of a previous screening) were assessed. The variables were varied according to a central composite design and the models obtained enabled evaluation and weighting of the effects of the independent variables on the free fatty acids (FFAs) and volatile profiles in pork fat based medium. The polynomial models showed the levels temperature, water activity of the pork fat based system and yeast strain inoculation were able to maximize the release of specific FFAs or molecules of sensory importance.     

The effect of substrate on mycotoxin production of selected Penicillium strains

Analytical methods are presented for detecting simultaneously 11 fungal metabolites (aflatoxins B1, B2, G1 and G2, citrinin, cyclopiazonic acid, mycophenolic acid, ochratoxin A, penicillic acid, penitrem A and roquefortine C) on different matrices. The methods were applied to determine the mycotoxins produced by different Penicillium crustosum, Penicillium nordicum and Penicillium verrucosum strains on yeast extract sucrose (YES) agar and cheese and bread analogues and are based on high-performance liquid chromatography (HPLC) and photodiode array detection (PDA). The growth substrate had a distinctive effect on the mycotoxin production ability of the fungi examined. The P. crustosum strains produced roquefortine C on all the substrates, with the highest amounts being detected on the cheese analogue. Penitrem A was synthesised on the cheese analogue only. The strains of P. verrucosum produced exclusively citrinin on YES, but both ochratoxin A and citrinin were detected in considerable amounts on the bread analogue. On the bread, toxin profiles varied significantly between the individual P. verrucosum strains. The cheese analogue was not favourable for the mycotoxin production of this species. The growth substrate had the least effect on the toxin production of the P. nordicum strains, which synthesised ochratoxin A in moderate amounts on all three media.     

Synergistic effect of fungicides on resistant strains of Penicillium italicum and Penicillium digitatum

In vitro and in vivo tests were carried out to study the inhibitory effect of mixtures of sodium orthophenylphenate, methyl-1-(butyl-carbamoyl)-2-benzimidazole ('Benomyl') and sodium orthophenylphenate, 2-(4-thiazolil)-benzimidazole ('Thiabendazole') on growth of strains of Penicillium italicum and P. digitatum resistant to sodium orthophenylphenate, 'Benomyl' and 'Thiabendazole'. 'In vitro' tests showed a synergistic effect of the mixtures; 10 ppm of sodium orthophenylphenate and 10 ppm of the benzimidazolic compound in most cases resulted in complete growth inhibition. Navel oranges inoculated with P. italicum spores showed no damage after 7 days of incubation at 25 degrees C and 80-90% relative humidity when treated with a mixture of 100 ppm of sodium orthophenylphenate and 100 ppm of Thiabendazole.     

Molecular monitoring of environmental conditions influencing the induction of ochratoxin A biosynthesis genes in Penicillium nordicum

A real-time polymerase chain reaction (PCR) system specific for the ochratoxin A polyketide synthase gene (otapksPN) of Penicillium nordicum has been used to analyze environmental conditions, influencing the induction of that key gene of the ochratoxin A biosynthetic pathway. Generally, the induction of that gene coincides very well with the biosynthesis of ochratoxin A, demonstrating that its induction can be used as a molecular signal to monitor ochratoxin A production. It could be shown, that the expression of the otapksPN gene is greatly dependent on the media used. In YES medium expression is highest, followed by minimal medium which support ochratoxin A production and minimal medium which suppresses ochratoxin A production. The amount of ochratoxin A produced shows the same tendency. The amount produced is highest on YES medium and decreases successively to the two minimal media. The system was also used to determine the influence of environmental parameters like temperature, pH and NaCl concentration on the expression of the otapksPN gene and on ochratoxin A production in parallel. It could be shown that under acidic conditions, below pH 5.0, the expression of the otapksPN gene as well as the ochratoxin A concentration were reduced. In case of salt concentration again both measures coincide, having both highest values at increasing NaCl concentrations. In case of the temperature, however, expression of the otapksPN gene was uncoupled to ochratoxin A production. The expression was high at all temperatures tested, however, clear differences in the biosynthesis of ochratoxin A by P. nordicum could be observed at the different temperatures, showing highest production at 25 degrees C. The importance of these data are discussed with reference to the natural habitat of P. nordicum.     

Inhibitory effects of eugenol and thymol on Penicillium citrinum strains in culture media and cheese

In the present work we studied the antifungal effect of eugenol and thymol on the growth and production of citrinin from Penicillium citrinum (NRRL 2274 and NRRL 2269) in culture media and in different Spanish cheeses (Arzúa-Ulloa, Cebreiro and San Simón). The rate of growth was assessed by measuring colony diameters and the production of citrinin was measured using a rapid semi-quantitative fluorometric technique confirmed by RP-HPLC. A stronger inhibitory effect of eugenol than thymol was evident. 200 microg/ml of eugenol in solid culture medium increased the lag time of growth up to 9 days, and decreased the rate of colony growth. In liquid medium, a complete inhibition of fungal growth was observed. By contrast, thymol in the liquid culture medium only affected the growth rate. In Arzúa-Ulloa cheese, 200 microg/ml of eugenol fully inhibited fungal growth, while in Cebreiro cheese no effect was observed for this compound. Regarding the capacity to inhibit mycotoxin production 100 microg/ml eugenol delayed citrinin production until the sixth day, after which a limiting effect persisted. In Arzúa-Ulloa cheese, no citrinin was detected at a concentration of 150 microg/ml of eugenol, but citrinin was detected after 5 days in the case of thymol at the same concentration. In Cebreiro cheese, neither eugenol nor thymol prevented the production of citrinin at the concentrations applied.     

The metabolism of fatty acids, methyl ketones and secondary alcohols by Penicillium roqueforti in blue cheese slurries.

A slurry system in which Blue cheese flavour was produced in six days was developed and used to study the metabolism of fatty acids, methyl ketones and secondary alcohols by Penicillium roqueforti. It was found that fatty acids inhibited lipolysis but that far greater quantities of methyl ketones were produced when fatty acids were added to the slurries than when fatty acids were produced by the lipolytic activity of P. roqueforti. Methyl ketones and secondary alcohols added to Blue cheese slurries in quantities ten times as great as those found in mature Blue cheese were broken down within 7 days And the presence of fatty acids did not appreciably inhibit this breakdown.