Characterization of Yeasts Isolated from Parmigiano Reggiano Cheese Natural Whey Starter: From Spoilage Agents to Potential Cell Factories for Whey Valorization

Whey is the main byproduct of the dairy industry and contains sugars (lactose) and proteins (especially serum proteins and, at lesser extent, residual caseins), which can be valorized by the fermentative action of yeasts. In the present study, we characterized the spoilage yeast population inhabiting natural whey starter (NWS), the undefined starter culture of thermophilic lactic acid bacteria used in Parmigiano Reggiano (PR) cheesemaking, and evaluated thermotolerance, mating type, and the aptitude to produce ethanol and bioactive peptides from whey lactose and proteins, respectively, in a selected pool of strains. PCR-RFLP assay of ribosomal ITS regions and phylogenetic analysis of 26S rDNA D1/D2 domains showed that PR NWS yeast population consists of the well-documented Kluyveromyces marxianus, as well as of other species (Saccharomyces cerevisiae, Wickerhamiella pararugosa, and Torulaspora delbrueckii), with multiple biotypes scored within each species as demonstrated by (GTG)5-based MSP-PCR. Haploid and diploid K. marxianus strains were identified through MAT genotyping, while thermotolerance assay allowed the selection of strains suitable to grow up to 48 °C. In whey fermentation trials, one thermotolerant strain was suitable to release ethanol with a fermentation efficiency of 86.5%, while another candidate was able to produce the highest amounts of both ethanol and bioactive peptides with potentially anti-hypertensive function. The present work demonstrated that PR NWS is a reservoir of ethanol and bioactive peptides producer yeasts, which can be exploited to valorize whey, in agreement with the principles of circularity and sustainability.

Changes in the Population of Schizosaccharomyces japonicus in Carissa edulis Vahl (Simple-spined Carissa) Juice Treated with Extracts of Citrus aurantifolia Christm. (lime) and Citrus limon Burm F. (lemon) Peels as Natural Preservatives during Storage

Aim: This study was aimed at evaluating the changes in the population of Schizosaccharomyces japonicus in locally produced simple-spine Carissa juice and the effects of lemon peel, lime peel and combination of lime and lemon peels extract as preservatives on the juice samples. Methodology: Fruit juice produced was treated with different concentrations of citrus peels extracts (lemon, lime and lime+lemon) and their shelf lives were determined at room temperature for 30 days. The effects of the different preservatives on the pH of the juices were assessed and the Yeast count of the juice samples treated with the various concentrations of the citrus peels and the untreated samples were assessed using standard microbiological methods. The sugar fermentation and assimilation test of the isolate was also determined. Results: Yeast population increased in the juice samples that were without treatments (control) from 5.6×103±200 to 1.52×104±200 cfuml-1 indicating significant increase (p≥0.05). Treated juice samples showed significant decrease (p≤0.05) in yeast population in the order of 300>200>100 mg/ml of the natural preservatives. Juice samples treated with highest concentration (300 mg/ml) of combined lime+lemon peels recorded the least colony forming units of 2.0×103±100 ml-1 during the storage period. This showed that the highest concentration (300 mg/ml) of combined lime+lemon peels had more effects on yeast load reduction in the Carissa juice. The results of the preservative properties of the citrus peels revealed the combined preservative (lime+lemon) as the best among the individual preservatives of lime and lemon. The sugar fermentation and assimilation tests showed that Schizosaccharomyces japonicus is a good fermenter of most sugars and can also assimilate sugars for its growth. The pH (3.40±0.01 and 4.09±0.01) of the treated fruit juices were within the acidic ranges that support the growth of yeast cells in culture. Conclusion: From the findings, organic extracts of citrus peels can be used to extend the shelf-life of Carissa juice for up to three weeks.

INVESTIGATION OF THE IMPACT OF THE THERMOSONICATION OPERATION ON DOOGH QUALITY AND ON THE SURVIVAL OF KLUYVEROMYCES MARXIANUS

Doogh is an Iranian drink produced from yoghurt. Swelling is an undesirable change of Doogh, usually caused by Kluyveromyces marxianus. The aim of this study was the investigation of the effect of the thermosonication process on Kluyveromyces marxianus survival as well as the determination of the impact of this process on other quality parameters (viscosity and serum separation). For this reason, Kluyveromyces marxianus (about 105 cells per milliliter) inoculated into Doogh samples, thereafter samples being exposed to various thermosonication process conditions. Results showed that the thermosonication operation resulted in the reduction of the yeast population of Doogh samples (p <0.05). The lowest yeast population was observed in the sample subjected to acoustic density=1w/ml, T= 60°C and t=30min (1.7±0.01CFU/ml). Other operation conditions also had impact on yeast survival (p <0.05). Thermosonication also had significant effects on the viscosity of the Doogh samples. Serum separation of samples was also measured and observation implied changing serum separation magnitude between different samples in comparison with the control sample (p <0.05).

Viability-PCR Allows Monitoring Yeast Population Dynamics in Mixed Fermentations Including Viable but Non-Culturable Yeasts

The use of controlled mixed inocula of Saccharomyces cerevisiae and non-Saccharomyces yeasts is a common practice in winemaking, with Torulaspora delbrueckii, Lachancea thermotolerans and Metschnikowia pulcherrima being the most commonly used non-Saccharomyces species. Although S. cerevisiae is usually the dominant yeast at the end of mixed fermentations, some non-Saccharomyces species are also able to reach the late stages; such species may not grow in culture media, which is a status known as viable but non-culturable (VBNC). Thus, an accurate methodology to properly monitor viable yeast population dynamics during alcoholic fermentation is required to understand microbial interactions and the contribution of each species to the final product. Quantitative PCR (qPCR) has been found to be a good and sensitive method for determining the identity of the cell population, but it cannot distinguish the DNA from living and dead cells, which can overestimate the final population results. To address this shortcoming, viability dyes can be used to avoid the amplification and, therefore, the quantification of DNA from non-viable cells. In this study, we validated the use of PMAxx dye (an optimized version of propidium monoazide (PMA) dye) coupled with qPCR (PMAxx-qPCR), as a tool to monitor the viable population dynamics of the most common yeast species used in wine mixed fermentations (S. cerevisiae, T. delbrueckii, L. thermotolerans and M. pulcherrima), comparing the results with non-dyed qPCR and colony counting on differential medium. Our results showed that the PMAxx-qPCR assay used in this study is a reliable, specific and fast method for quantifying these four yeast species during the alcoholic fermentation process, being able to distinguish between living and dead yeast populations. Moreover, the entry into VBNC status was observed for the first time in L. thermotolerans and S. cerevisiae during alcoholic fermentation. Further studies are needed to unravel which compounds trigger this VBNC state during alcoholic fermentation in these species, which would help to better understand yeast interactions.