Analysis of phthalate plasticizer migration from PVDC packaging materials to food simulants using molecular dynamics simulations and artificial neural network.

Based on the experimental data of gas chromatography-mass spectrometry, an improved artificial neural network was first established to predict the migration of 2-ethylhexyl phthalate (DEHP) plasticizer from poly(vinylidene chloride) (PVDC) into food simulants (ie., heptane, ethanol and water). The sensitivity analysis indicated that temperature acted as a crucial factor influencing the migration values of DEHP. Then, a combined experimental and molecular dynamic (MD) simulation was performed to understand the migration kinetics and the mechanism of DEHP. Hansen solubility parameters of three component (δd, δp, δh) were simplified into two-component solubility parameters (δvdW, δe), and the tuple was successfully applied to describe the interactions between PVDC and food simulants. The MD results showed that high interaction energy and fractional free volume in PVDC/DEHP/food simulant systems accelerated the migration of DEHP. These fundamental studies would provide significant insights into the migration of environmental contaminants.

Biocontrol of Penicillium griseofulvum to reduce cyclopiazonic acid contamination in dry-fermented sausages.

Dry-fermented sausages are very appreciated by consumers. The environmental conditions during its ripening favor colonization of their surface by toxigenic molds. These molds contribute to the development of sensory characteristics; however, some of them could produce mycotoxins such as cyclopiazonic acid (CPA). CPA is mainly produced by Penicillium commune and Penicillium griseofulvum which have been found in dry-cured meat products. Thus, strategies to prevent the CPA contamination in dry-fermented sausages are needed. The objective of this work was to evaluate the ability of P. griseofulvum to produce CPA in dry-fermented sausage during its ripening as well as to test different strategies to prevent CPA production. The ability of PgAFP antifungal protein-producing Penicillium chrysogenum, Debaryomyces hansenii and Pediococcus acidilactici for inhibiting CPA production by P. griseofulvum was tested on dry-fermented sausage-based medium. Only P. chrysogenum inhibited the CPA production, so this mold was co-inoculated with P. griseofulvum on sausages whose ripening was performed at low temperature. CPA reached around 800 ng/g in the control batch, being reduced to 20 ng/g by the presence of P. chrysogenum. This work demonstrates the risk posed by CPA on dry-fermented sausages, and provides a successful strategy to prevent this hazard.

Fourier Transform Near-Infrared Spectroscopy and Chemometrics To Predict Zygosacchromyces rouxii in Apple and Kiwi Fruit Juices.

This study investigated the capability of near-infrared spectroscopy (NIRS) to predict the concentration of Zygosaccharomyces rouxii in apple and kiwi fruit juices. The yeast was inoculated in fresh kiwi fruit juice ( n = 68), reconstituted kiwi juice ( n = 85), and reconstituted apple juice ( n = 64), followed by NIR spectra collection and plate counting. A principal component analysis indicated direct orthogonal signal correction preprocessing was suitable to separate spectral samples. Parameter optimization algorithms increased the performance of support vector machine regression models developed in a single variety juice system and a multiple variety juice system. Single variety juice models achieved accurate prediction of Z. rouxii concentrations, with the limit of quantification at 3 to 15 CFU/mL ( R2 = 0.997 to 0.999), and the method was also feasible for Hanseniaspora uvarum and Candida tropicalis. The best multiple variety juice model obtained had a limit of quantification of 237 CFU/mL ( R2 = 0.961) for Z. rouxii. A Bland-Altman analysis indicated good agreement between the support vector machine regression model and the plate counting method. It suggests that NIRS can be a high-throughput method for prediction of Z. rouxii counts in kiwi fruit and apple juices.

Identification of acetic acid bacteria in traditionally produced vinegar and mother of vinegar by using different molecular techniques.

Culture-dependent and culture-independent methods were combined for the investigation of acetic acid bacteria (AAB) populations in traditionally produced vinegars and mother of vinegar samples obtained from apple and grape. The culture-independent denaturing gradient gel electrophoresis (DGGE) analysis, which targeted the V7-V8 regions of the 16S rRNA gene, showed that Komagataeibacter hansenii and Komagataeibacter europaeus/Komagataeibacter xylinus were the most dominant species in almost all of the samples analyzed directly. The culture-independent GTG5-rep PCR fingerprinting was used in the preliminary characterization of AAB isolates and species-level identification was carried out by sequencing of the 16S rRNA gene, 16S-23S rDNA internally transcribed to the spacer (ITS) region and tuf gene. Acetobacter okinawensis was frequently isolated from samples obtained from apple while K. europaeus was identified as the dominant species, followed by Acetobacter indonesiensis in the samples originating from grape. In addition to common molecular techniques, real-time PCR intercalating dye assays, including DNA melting temperature (Tm) and high resolution melting analysis (HRM), were applied to acetic acid bacterial isolates for the first time. The target sequence of ITS region generated species-specific HRM profiles and Tm values allowed discrimination at species level.

Yeast diversity and dynamics in the production processes of Norwegian dry-cured meat products.

This study investigate the diversity and dynamics of yeasts in the production processes of one unsmoked and two smoked dry-cured meat products of a Norwegian dry-cured meat production facility. A longitudinal observational study was performed to collect 642 samples from the meat, production materials, room installations and indoor and outdoor air of the production facility. Nutrient rich agar media were used to isolate the yeasts. Morphologically different isolates were re-cultivated in their pure culture forms. Both classical and molecular methods were employed for species identification. Totally, 401 yeast isolates belonging to 10 species of the following six genera were identified: Debaryomyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus and Sporidiobolus. Debaryomyces hansenii and Candida zeylanoides were dominant and contributed by 63.0% and 26.4% respectively to the total isolates recovered from both smoked and unsmoked products. The yeast diversity was higher at the pre-salting production processes with C. zeylanoides being the dominant. Later at the post-salting stages, D. hansenii occurred frequently. Laboratory studies showed that D. hansenii was more tolerant to sodium chloride and nitrite than C. zeylanoides. Smoking seems to have a killing or a temporary growth inhibiting effect on yeasts that extend to the start of the drying process. Yeasts were isolated only from 31.1% of the environmental samples. They belonged to six different species of which five of them were isolated from the meat samples too. Debaryomyces hansenii and Rhodotorula glutinis were dominant with a 62.6% and 22.0% contribution respectively. As none of the air samples contained D. hansenii, the production materials and room installations used in the production processes were believed to be the sources of contamination. The dominance of D. hansenii late in the production process replacing C. zeylanoides should be considered as a positive change both for the quality and safety of the products, as C. zeylanoides has been documented as an emerging pathogen.

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.

Application of whole genome amplification and quantitative PCR for detection and quantification of spoilage yeasts in orange juice.

Small cell numbers in complex food matrices and undefined PCR inhibitors often limit detection and identification of DNA species by molecular techniques. Thus in many industrial situations enrichment growths are performed. However, growth speed of different species in complex microbial mixtures in defined media is in most cases different, thus final results do not always reflect the starting situation. We tested DNA-strand displacement whole genome amplification as a possible substitute of enrichment growth. Using whole genome amplification on orange juice contaminated with Saccharomyces cerevisiae, we lowered detection level from 10(6) down to 10(2) cfu/ml. Whole genome amplification showed to be linear (R=0.992) and the relative yeast DNA copy number compared to other DNA templates did not change thus allowing quantitative estimation of initial contamination by quantitative PCR. Using melting point analysis, we were able to distinguish between the PCR products of the 5.8S-ITS region, obtained with universal primers from pure cultures of S. cerevisiae and Hanseniaspora uvarum, two major spoilage yeasts in orange juice and forming part of wine microbiota during fermentation. However, in mixed-contaminated samples, identification of both species was hampered by preferential appearance of the melting peak coinciding with H. uvarum, except when S. cerevisiae was the dominating species. Application of whole genome amplification did not prevent the preferential detection of H. uvarum. This handicap was resolved by applying an enrichment procedure up to saturation after which the melting peak of both species could clearly be identified.

The effect of pyrimethanil on the growth of wine yeasts.

AIMS: The toxicity of the fungicide pyrimethanil on the growth of wine yeasts was evaluated using in vivo and in vitro experimentation. METHODS AND RESULTS: The effect of pyrimethanil in the must was studied during the spontaneous wine fermentation of three consecutive vintages and by the cultivation of Hanseniaspora uvarum and Saccharomyces cerevisiae yeasts in a liquid medium. The residues of the fungicide were measured using gas chromatography-mass spectrometry system and the sugar concentration in the must using HPLC-RI. Molecular and standard methods were used for identifying the yeast species. Although the pyrimethanil residues in grapes were below the maximum residue limits, they significantly affected the reduced utilization of sugars in the first days of fermentation. Its residues controlled the growth of H. uvarum during the fermentation and during in vitro cultivation as well. CONCLUSIONS: The fungicide pyrimethanil had an effect on the course and successful conclusion of spontaneous wine fermentation that was correlated with the initial concentration of yeasts in the must. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of pyrimethanil on the indigenous mixed yeast flora in fermenting must was investigated for the first time. The results showed that its residues might play an important role in the growth and succession of yeast during spontaneous wine fermentation.

[Research on QSPR for n-octanol-water partition coefficients of organic compounds based on genetic algorithms-support vector machine and genetic algorithms-radial basis function neural networks].

A modified method to develop quantitative structure-property relationship (QSPR) models of organic compounds was proposed based on genetic algorithm (GA) and support vector machine (SVM) (GA-SVM). GA was used to perform the variable selection, and SVM was used to construct QSPR models. GA-SVM was applied to develop the QSPR models for n-octanol-water partition coefficients ( Kow) of 38 typical organic compounds in food industry. 5 descriptors (molecular weights, Hansen polarity, boiling point, percent oxygen and percent hydrogen) were selected in the QSPR model. The coefficient of multiple determination (R2), the sum of squares due to error (SSE) and the root mean squared error (RMSE) values between the measured values and predicted values of the model developed by GA-SVM are 0.999, 0.048 and 0.036, respectively, indicating good predictive capability for lgKow values of these organic compounds. Based on leave-one-out cross validation, the QSPR model constructed by GA-SVM showed good robustness (SSE = 0.295, RMSE = 0.089, R2 = 0.995). Moreover, the models developed by GA-SVM were compared with the models constructed by genetic algorithm-radial basis function neural network (GA-RBFNN) and linear method. The models constructed by GA-SVM show the optimal predictive capability and robustness in the comparison, which illustrates GA-SVM is the optimal method for developing QSPR models for lgKow values of these organic compounds.

Dynamics and characterization of yeasts during ripening of typical Italian dry-cured ham.

The evolution of the yeast population during manufacturing and ripening of dry-cured Parma ham was investigated. Contamination levels ranged from 10(5) to 10(7) cfu/g on muscle surface, 10(4) to 10(6) cfu/g on covering fat and exceeded 10(7) cfu/g on spreadable fat mince ("sugna"). Two hundred and sixty one yeast isolates underwent identification test, showing that the predominant species of yeast population during the whole maturing process were Debaryomyces hansenii, Candida zeylanoides, Debaryomyces maramus, and to a lesser extent, Candida famata and Hyphopichia burtonii. The species Candida catenulata, Candida guilliermondii, Candida edax and other genera like Cryptococcus and Wingea were occasionally found. The yeast counts and species distribution changed according to the stage of processing and to the ham sampling location. At the end of the cold phase, the washing procedure was effective in lowering the yeast count in muscle and fat surface layers, but during the next ageing stages, yeast colonization of unskinned ham muscle increased again, though species distribution changed if compared to previous manufacturing phases. The ripening steps taken into account from the end of the cold phase to the final outcome, were always characterized by more than one yeast species, suggesting that yeasts other than Debaryomyces spp. could play a remarkable role on the sensory and safety properties of typical Italian dry-cured ham.

Molecular monitoring of spoilage yeasts during the production of candied fruit nougats to determine food contamination sources.

In the present work, we have analysed the yeast microbiota present in a manufacturing plant of candied fruits and nougats. Four yeasts species (Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Sporobolomyces roseus, and Debaryomyces hansenii) and a filamentous fungi (Nectria mauriiticola) were identified according to restriction analysis of 5.8S-ITS rDNA. These identifications were subsequently confirmed by sequencing the D1/D2 domain of the 26S rRNA gene. Z. rouxii and Z. bailii were isolated at high frequency along the whole manufacturing process. Since food alteration by Z. bailii and Z. rouxii is the cause of important economic losses for the food industry, there is a need for differentiating yeasts at the strain level as an essential part of quality control programs in this industry. For this purpose, we have tested the performance of three molecular techniques (RFLP mtDNA, RAPD-PCR, and microsatellite with (GAC)5 and (GTG)5 primers) to differentiate strains belonging to these two Zygosaccharomyces species. Those techniques with the best discriminatory power were applied to differentiate Zygosaccharomyces species isolates. The results of this analysis indicate that one strain of Z. bailii and two strains of Z. rouxii were involved in the spoilage of candied fruits. Moreover, the Z. bailii strain was also present in the spoiled nougat, hence being responsible of this alteration.

A beta-glucuronidase-based agar medium for the differential detection of the yeast Debaryomyces hansenii from foods.

A selective and differential solid medium, Debaryomyces differential medium (DDM), was used for the isolation of Debaryomyces hansenii. This medium is formulated to allow detection of the beta-glucuronidase enzyme using the chromogenic substrate magenta-glucuro.CHA (5Br-6Cl-3indolyl-beta-D-glucuronide, cyclohexylammonium salt). Of the more than 120 microorganisms tested, including yeasts, bacteria, and a filamentous fungus, only D. hansenii produced violet colonies, thus permitting its easy discrimination from other organisms. When quality assessment tests were performed, optimal productivity and selectivity were obtained for D. hansenii. The medium was also satisfactory when used to test naturally contaminated food products.