Competitiveness of three biocontrol candidates against ochratoxigenic Penicillium nordicum under dry-cured meat environmental and nutritional conditions.

The environmental conditions during the ripening of dry-cured meats and their nutritional composition promote the colonisation of their surface by Penicillium spp., including P. nordicum producer of ochratoxin A (OTA). The objective of this work was to study the competitiveness of three potential biocontrol candidates (Debaryomyces hansenii FHSCC 253H, Enterococcus faecium SE920 and Penicillium chrysogenum CECT, 20922) against the ochratoxigenic P. nordicum FHSCC4 under environmental and nutritional conditions simulating the ripening of dry-cured meat products. For this, the nutritional utilisation pattern, niche overlap index (NOI), interactions by dual-culture assays and OTA production were determined. The number of carbon sources (CSs) metabolised depended on the microorganism and the interacting water activity (aw) x temperature conditions. The number of CSs utilised by both filamentous fungi was quite similar and higher than those utilised by D. hansenii and E. faecium. The yeast isolate metabolised a number of CSs much larger than the bacterium. The NOI values showed that, in general, P. nordicum nutritionally dominated E. faecium and D. hansenii regardless of the environmental conditions evaluated. The relationship between the toxigenic and non-toxigenic fungal isolates depended on the aw x temperature combinations, although in none of the conditions a dominance of P. nordicum was observed. According to the interaction assays, both D. hansenii and P. chrysogenum decreased the growth of P. nordicum. The effect of D. hansenii could be attributed to the production of some extra-cellular compounds, while the action of P. chrysogenum is likely related to nutritional competition. In addition, both P. chrysogenum and D. hansenii reduced the OTA levels produced by P. nordicum. The effect of the yeast was more pronounced decreasing the concentration of OTA at quantities lower than the limit established by the Italian legislation. Therefore, P. chrysogenum and D. hansenii can be suggested as biocontrol candidates in the manufacture of dry-cured meat products.

Effect of different inoculation strategies of selected yeast and LAB cultures on Conservolea and Kalamàta table olives considering phenol content, texture, and sensory attributes.

BACKGROUND: The effects were studied of different inoculation strategies for selected starters -yeasts and lactic acid bacteria (LAB) - used for the fermentation process of two Greek olive cultivars, Conservolea and Kalamàta. The LAB strains applied were Leuconostoc mesenteroides K T5-1 and L. plantarum A 135-5; the selected yeast strains were S. cerevisiae KI 30-16 and Debaryomyces hansenii A 15-44 for Kalamàta and Conservolea olives, respectively. RESULTS: Table olive fermentation processes were monitored by performing microbiological analyses, and by monitoring changes in pH, titratable acidity and salinity, sugar consumption, and the evolution of volatile compounds. Structural modifications occurring in phenolic compounds of brine were investigated during the fermentation using liquid chromatography / diode array detection / electrospray ion trap tandem mass spectrometry (LC/DAD/ESI-MSn ) and quantified by high-performance liquid chromatography (HPLC) using a diode array detector. Phenolic compounds in processed Kalamàta olive brines consisted of phenolic acids, verbascoside, caffeoyl-6-secologanoside, comselogoside, and the dialdehydic form of decarboxymethylelenolic acid linked to hydroxytyrosol, whereas oleoside and oleoside 11-methyl ester were identified only in Conservolea olive brines. CONCLUSION: Volatile profile and sensory evaluation revealed that the 'MIX' (co-inoculum of yeast and LAB strain) inoculation strategy led to the most aromatic and acceptable Kalamàta olives. For the Conservolea table olives, the 'YL' treatment gave the most aromatic and the overall most acceptable product. © 2019 Society of Chemical Industry.

Inhibitory effect of four novel synthetic peptides on food spoilage yeasts.

The spoilage of foods caused by the growth of undesirable yeast species is a problem in the food industry. Yeast species such as Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii, Kluyveromyces lactis and Saccharomyces cerevisiae have been encountered in foods such as high sugar products, fruit juices, wine, mayonnaise, chocolate and soft drinks. The demand for new methods of preservations has increased because of the negative association attached to chemical preservatives. The sequence of a novel short peptide (KKFFRAWWAPRFLK-NH2) was modified to generate three versions of this original peptide. These peptides were tested for the inhibition of the yeasts mentioned above, allowing for the better understanding of their residue modifications. The range of the minimum inhibitory concentration was between 25 and 200 µg/mL. Zygosaccharomyces bailii was the most sensitive strain to the peptides, while Zygosaccharomyces rouxii was the most resistant. Membrane permeabilisation was found to be responsible for yeast inhibition at a level which was a two-fold increase of the MIC (400 µg/mL). The possibility of the production of reactive oxygen species was also assessed but was not recognised as a factor involved for the peptides' mode of action. Their stability in different environments was also tested, focusing on high salt, pH and thermal stability. The newly designed peptides showed good antifungal activity against some common food spoilage yeasts and has been proven effective in the application in Fanta Orange. These efficient novel peptides represent a new source of food preservation that can be used as an alternative for current controversial preservatives used in the food industry.

Optimization of fermentation-relevant factors: A strategy to reduce ethanol in red wine by sequential culture of native yeasts.

Current consumer preferences are determined by well-structured, full-bodied wines with a rich flavor and with reduced alcohol levels. One of the strategies for obtaining wines with reduced ethanol content is sequential inoculation of non-Saccharomyces and Saccharomyces cerevisiae yeasts. However, different factors affect the production of metabolites like ethanol, glycerol and acetic acid by inoculated yeasts. In order to obtain low alcohol wines without quality loss, the aims of our study were: i) to determine optimum conditions (fermentation temperature and time of permanence and initial inoculum size of the non-Saccharomyces population at the beginning of the process, prior to inoculation with S. cerevisiae); ii) to validate the optimized factors; and iii) to assess sensory quality of the wines obtained after validation. Two combinations of yeasts were used in this study: Hanseniaspora uvarum BHu9/S. cerevisiae BSc114 and Candida membranaefaciens BCm71/S. cerevisiae BSc114. Optimization of three fermentation factors that affect to non-Saccharomyces yeasts prior to S. cerevisiae inoculation was carried out using a Box-Behnken experimental design. Applying the models constructed by Response Surface Methodology, the lowest ethanol production by H. uvarum BHu9/S. cerevisiae BSc114 co-culture was obtained when H. uvarum BHu9 was inoculated 48 h 37 min prior to S. cerevisiae inoculation, at a fermentation temperature of 25 °C and at an initial inoculum size of 5 × 106 cells/mL. Lowest alcohol production with C. membranaefaciens BCm71/S. cerevisiae BSc114 was observed when C. membranaefaciens BCm71 was inoculated 24 h 15 min prior to S. cerevisiae at a fermentation temperature of 24.94 °C and at an initial inoculum size of 2.72 × 106 cells/mL. The optimized conditions of the two co-cultures were subsequently submitted to lab-scale validation. Both proposed strategies yielded ethanol levels that were significantly lower than control cultures (S. cerevisiae). Wines fermented with non-Saccharomyces/Saccharomyces co-cultures under optimized conditions were also associated with higher aromatic complexity characterized by the presence of red fruit aromas, whereas wines obtained with S. cerevisiae BSc114 were described by parameters linked with high ethanol levels.

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.

Wine aroma response to different participation of selected Hanseniaspora uvarum in mixed fermentation with Saccharomyces cerevisiae.

Wine aroma response to a selected Hanseniaspora uvarum Yun268 strain was investigated using different inoculation strategies with commercial Saccharomyces cerevisiae yeast, namely, simultaneous fermentation (SiF), sequential fermentation (SeF), S. cerevisiae fermentation treated with extracellular extract of H. uvarum (EE), and pure S. cerevisiae fermentation (PF). Contributive volatiles in the perception of enhanced aroma traits were uncovered by partial least-squares regression. Results showed that controlled inoculation resulted into different amounts of H. uvarum Yun268, which distinctively affected the chemical and sensory profiles of wines. The concentration of aromatic compounds could be increased by H. uvarum Yun268 yeasts via high levels of ß-glucosidase activity and fatty acids. Terpenes, C13-norisoprenoids, acetate esters, ethyl esters, and fatty acids served as the impact volatiles that contributed to the enhanced aroma traits. SiF specifically increased the contents of C13-norisoprenoids, terpenes, and ethyl esters, while EE enhanced varietal volatile content rather than those of fermentative ones. However, excessive H. uvarum Yun268 in sequential inoculation elevated the concentrations of acetate esters and volatile phenols, triggering nail polish odor in Cabernet Sauvignon red wines.

Primary souring: A novel bacteria-free method for sour beer production.

In the beverage fermentation industry, especially at the craft or micro level, there is a movement to incorporate as many local ingredients as possible to both capture terroir and stimulate local economies. In the case of craft beer, this has traditionally only encompassed locally sourced barley, hops, and other agricultural adjuncts. The identification and use of novel yeasts in brewing lags behind. We sought to bridge this gap by bio-prospecting for wild yeasts, with a focus on the American Midwest. We isolated 284 different strains from 54 species of yeast and have begun to determine their fermentation characteristics. During this work, we found several isolates of five species that produce lactic acid and ethanol during wort fermentation: Hanseniaspora vineae, Lachancea fermentati, Lachancea thermotolerans, Schizosaccharomyces japonicus, and Wickerhamomyces anomalus. Tested representatives of these species yielded excellent attenuation, lactic acid production, and sensory characteristics, positioning them as viable alternatives to lactic acid bacteria (LAB) for the production of sour beers. Indeed, we suggest a new LAB-free paradigm for sour beer production that we term "primary souring" because the lactic acid production and resultant pH decrease occurs during primary fermentation, as opposed to kettle souring or souring via mixed culture fermentation.

Evaluation of a cross contamination model describing transfer of Salmonella spp. and Listeria monocytogenes during grinding of pork and beef.

In a previous study, a model was developed to describe the transfer and survival of Salmonella during grinding of pork (Møller, C.O.A., Nauta, M.J., Christensen, B.B., Dalgaard, P., Hansen, T.B., 2012. Modelling transfer of Salmonella typhimurium DT104 during simulation of grinding of pork. Journal of Applied Microbiology 112 (1), 90-98). The robustness of this model is now evaluated by studying its performance for predicting the transfer and survival of Salmonella spp. and Listeria monocytogenes during grinding of different types of meat (pork and beef), using two different grinders, different sizes and different numbers of pieces of meats to be ground. A total of 19 grinding trials were collected. Acceptable Simulation Zone (ASZ), visual inspection of the data, Quantitative Microbiological Risk Assessment (QMRA), as well as the Total Transfer Potential (TTP) were used as approaches to evaluate model performance and to access the quality of the cross contamination model predictions. Using the ASZ approach and considering that 70% of the observed counts have to be inside a defined acceptable zone of ±0.5 log10CFU per portion, it was found that the cross contamination parameters suggested by Møller et al. (2012) were not able to describe all 19 trials. However, for each of the collected grinding trials, the transfer event was well described when fitted to the model structure proposed by Møller et al. (2012). Parameter estimates obtained by fitting observed trials performed at different conditions, such as size and number of pieces of meat to be ground, may not be applied to describe cross contamination of unlike processing. Nevertheless, the risk estimates, as well as the TTP, revealed that the risk of disease may be reduced when the grinding of meat is performed in a grinder made of stainless steel (for all surfaces in contact with the meat), using a well-sharpened knife and holding at room temperatures lower than 4°C.

Modelling and predicting the simultaneous growth of Escherichia coli and lactic acid bacteria in milk.

Modelling and predicting the simultaneous competitive growth of Escherichia coli and starter culture of lactic acid bacteria (Fresco 1010, Chr. Hansen, Hørsholm, Denmark) was studied in milk at different temperatures and Fresco inoculum concentrations. The lactic acid bacteria (LAB) were able to induce an early stationary state in E. coli The developed model described and tested the growth inhibition of E. coli (with initial inoculum concentration 10(3) CFU/mL) when LAB have reached maximum density in different conditions of temperature (ranging from 12 ℃ to 30 ℃) and for various inoculum sizes of LAB (ranging from approximately 10(3) to 10(7) CFU/mL). The prediction ability of the microbial competition model (the Baranyi and Roberts model coupled with the Gimenez and Dalgaard model) was first performed only with parameters estimated from individual growth of E. coli and the LAB and then with the introduced competition coefficients evaluated from co-culture growth of E. coli and LAB in milk. Both the results and their statistical indices showed that the model with incorporated average values of competition coefficients improved the prediction of E. coli behaviour in co-culture with LAB.

Development of species-specific primers for rapid identification of Debaryomyces hansenii.

In this work, we developed a specific PCR assay for Debaryomyces hansenii strains that uses a putative homologous PAD1 region (729 bp) present in this yeast species as a target. The amplification of this sequence with the D. hansenii specific primer pair (DhPADF/DhPADR) was found to be a rapid, specific and an affordable method enabling identification of D. hansenii from other yeast strains. Primers were tested in almost 100 strains, 49 strains from Type Culture Collection belonging to the genus Debaryomyces and to other yeast species commonly found in foods or related genera. These primers were able to discriminate between closely related species of Debaryomyces, such as Debaryomyces fabryi and Debaryomyces subglobosus, with a 100% detection rate for D. hansenii. Also, the method was tested in 45 strains from different foods. Results confirmed the specificity of the PCR method and detected two earlier misidentifications of D. hansenii strains obtained by RFLP analysis of the 5.8S ITS rDNA region. Subsequently we confirmed by sequencing the D1/D2 domain of 26S rDNA that these strains belonged to D. fabryi. We call attention in this work to the fact that the RFLPs of the 5.8S ITS rDNA profiles of D. hansenii, D. fabryi and D. subglobosus are the same and this technique will thus lead to incorrect identifications.

Monitoring of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris (synonym Candida zemplinina) populations during alcoholic fermentation by fluorescence in situ hybridization.

Various molecular approaches have been applied as culture-independent techniques to monitor wine fermentations over the last decade. Among them, those based on RNA detection have been widely used for yeast cell detection, assuming that RNA only exists in live cells. Fluorescence in situ hybridization (FISH) targeting intracellular rRNA is considered a promising technique for the investigation of wine ecology. For the present study, we applied the FISH technique in combination with epifluorescence microscopy and flow cytometry to directly quantify populations of Saccharomyces cerevisiae, Hanseniaspora uvarum, and Starmerella bacillaris during alcoholic fermentations. A new specific probe that hybridizes with eight species of Hanseniaspora genus and a second probe specific for Starm. bacillaris were designed, and the conditions for their application to pure cultures, mixed cultures, and wine samples were optimized. Single and mixed fermentations were performed with natural, concentrated must at two different temperatures, 15 °C and 25 °C. The population dynamics revealed that the Sacch. cerevisiae population increased to 10(7)-10(8)cells/ml during all fermentations, whereas H. uvarum and Starm. bacillaris tended to increase in single fermentations but remained at levels similar to their inoculations at 10(6)cells/ml in mixed fermentations. Temperature mainly affected the fermentation duration (slower at the lower temperature) but did not affect the population sizes of the different species. The use of these probes in natural wine fermentations has been validated.