Pulsed electric field processing as an alternative to sulfites (SO2) for controlling saccharomyces cerevisiae involved in the fermentation of Chardonnay white wine

The use of sulfites (SO2) for microbial control in the winemaking process is currently being questioned due to its potential toxicity. Pulsed Electric Fields (PEF) are capable of inactivating microorganisms at low temperatures, thus avoiding the negative effects of heat on food properties. In this study, the capacity of PEF technology for the decontamination of yeasts involved in the fermentation process of Chardonnay wine from a winery was evaluated. PEF treatments at 15 kV/cm of low (65 µs, 35 kJ/kg) and higher intensity (177 µs 97 kJ/kg) were selected for evaluating the microbial stability, physicochemical and volatile composition of wine. Even with the least intense PEF-treatment, Chardonnay wine remained yeast-free during 4 months of storage without sulfites. PEF-treatments did not affect the wine’s oenological parameters or its aroma during storage. This study, therefore, reveals the potential of PEF technology as an alternative to sulfites for the microbiological stabilization of wine

Sequential extraction of compounds of interest from yeast biomass assisted by pulsed electric fields

One strategy to reduce cost and improve feasibility of waste-yeast biomass valorization is to obtain a spectrum of marketable products rather than just a single one. This study explores the potential of Pulsed Electric Fields (PEF) for the development of a cascade process designed to obtain several valuable products from Saccharomyces cerevisiae yeast biomass. Yeast biomass was treated by PEF, which affected the viability of 50%, 90%, and over 99% of S. cerevisiae cells, depending on treatment intensity. Electroporation caused by PEF allowed access to the cytoplasm of the yeast cell without causing total breakdown of the cell structure. This outcome was an essential prerequisite to be able to perform a sequential extraction of several value-added biomolecules from yeast cells located in the cytosol and in the cell wall. After incubating yeast biomass previously subjected to a PEF treatment that affected the viability of 90% of cells for 24 h, an extract with 114.91 ± 2.86, 7.08 ± 0.64, and 187.82 ± 3.75 mg/g dry weight of amino acids, glutathione, and protein, respectively, was obtained. In a second step, the extract rich in cytosol components was removed after 24 h of incubation and the remaining cell biomass was re-suspended with the aim of inducing cell wall autolysis processes triggered by the PEF treatment. After 11 days of incubation, a soluble extract containing mannoproteins and pellets rich in β-glucans were obtained. In conclusion, this study proved that electroporation triggered by PEF permitted the development of a cascade procedure designed to obtain a spectrum of valuable biomolecules from S. cerevisiae yeast biomass while reducing the generation of waste

Defining winery processing conditions for the decontamination of must and wine spoilage microbiota by Pulsed Electric Fields (PEF)

This study investigated the PEF-resistance of Saccharomyces bayanus, Brettanomyces bruxellensis, Lactobacillus plantarum, and Oenococus oeni in must or wine under continuous PEF processing. Results showed the capacity of PEF to achieve 3.0-log10-cycles (CFU/mL) of inactivation of all the microorganisms under moderate conditions (< 155 kJ/kg). Developed tertiary models accurately predicted the effect of PEF parameters on microbial inactivation, and Monte Carlo simulation considered the variability of factors and the maximum assumable microbial load in the final treated product. Results showed that PEF-treatments at 15 kV/cm and 129 or 153 kJ/kg would ensure the adequate decontamination (< 10 CFU/mL) of spoilage microorganism in must or wine, respectively. Industrial relevance: PEF technology has been shown to achieve adequate levels of microbial inactivation (3-log10) in must and wine under industrial applicable processing parameters, making it a suitable alternative to SO2 or sterilizing filtration for microbial control in winemaking. Reductions of 3-log10 CFU/mL of must and wine microbiota were found by continuous flow PEF-processing at 15 to 25 kV/cm and 175 to 148 kJ/kg, parameters applicable at industrial scale at 1 ton/h

Organic-solvent-free extraction of carotenoids from yeast Rhodotorula glutinis by application of ultrasound under pressure

The extraction of Rhodotorula glutinis carotenoids by ultrasound under pressure (manosonication) in an aqueous medium has been demonstrated. The influence of treatment time, pressure, and ultrasound amplitude on R. glutinis inactivation and on the extraction of carotenoids was evaluated, and the obtained data were described mathematically. The extraction yields were lineal functions of those three parameters, whereas inactivation responded to a more complex equation. Under optimum treatment conditions, 82% of carotenoid content was recovered. Extraction of carotenoids in an aqueous medium was attributed to the capacity of ultrasound for cell disruption and emulsification. Cavitation caused the rupture of cell envelopes and the subsequent formation of small droplets of carotenoids surrounded by the phospholipids of the cytoplasmic membrane that would stabilize the emulsion. Analysis of the dispersed particle size of the extracts demonstrated that a fine, homogeneous emulsion was formed after treatment (average size: 230 nm; polydispersity <0.22). This research describes an innovative green process for extracting carotenoids from fresh biomass of R. glutinis in which only two unit operations are required: ultrasonic treatment, followed by a centrifugation step to discard cell debris. The extract obtained thanks to this procedure is rich in carotenoids (25 mg/L) and could be directly incorporated as a pigment in foods, beverages, and diet supplements; it can also be utilized as an ingredient in drugs or cosmetics

Post-incubation pH Impacts the Lipid Extraction Assisted by Pulsed Electric Fields from Wet Biomass of Auxenochlorella protothecoides

Pulsed electric field (PEF) treatment is a promising technology for efficient lipid extraction from microalgae. This study focusses on under-investigated processing parameters, such as media pH, pulse application, and incubation protocols. The lipid yield and electroporation level of PEF-treated Auxenochlorella protothecoides were determined at a medium pH of 3.0 and 5.0 under variation of the pre- or post-PEF incubation time and for split-dose treatments. Low energetic PEF treatments with 40 kV/cm and 1 μs pulses at 9.6 and 19.2 kJ/L were performed either in batch mode or in continuous flow. Post-PEF incubation significantly increased the shared electroporated cells (>60%) in medium pH 3.0, while no change was observed at pH 5.0. Split-dose PEF treatments at pH 5.0 caused significantly higher electroporation levels and lipid extraction yields than equivalent single-dose treatments. Results have shown that medium pH is critical in the final electroporation and lipid extraction yields of A. protothecoides and therefore should be considered in further studies

Post-incubation pH Impacts the Lipid Extraction Assisted by Pulsed Electric Fields from Wet Biomass of "Auxenochlorella protothecoides"

Pulsed electric field (PEF) treatment is a promising technology for efficient lipid extraction from microalgae. This study focusses on under-investigated processing parameters, such as media pH, pulse application, and incubation protocols. The lipid yield and electroporation level of PEF-treated Auxenochlorella protothecoides were determined at a medium pH of 3.0 and 5.0 under variation of the pre- or post-PEF incubation time and for split-dose treatments. Low energetic PEF treatments with 40 kV/cm and 1 μs pulses at 9.6 and 19.2 kJ/L were performed either in batch mode or in continuous flow. Post-PEF incubation significantly increased the shared electroporated cells (>60%) in medium pH 3.0, while no change was observed at pH 5.0. Split-dose PEF treatments at pH 5.0 caused significantly higher electroporation levels and lipid extraction yields than equivalent single-dose treatments. Results have shown that medium pH is critical in the final electroporation and lipid extraction yields of A. protothecoides and therefore should be considered in further studies

Aplicación de la tecnología de Pulsos eléctricos de Alto Voltaje (PEAV) para el control de los microorganismos alterantes del vino

La presencia de microorganismos alterantes durante la elaboración del vino es una de las principales problemáticas de la industria vinícola ya que genera numerosas pérdidas económicas, especialmente cuando se generan cambios sensoriales que afectan la calidad del vino final. Actualmente las técnicas que se aplican para su control son la ampliamente utilizada adicción de dióxido de azufre (SO2) y la filtración esterilizante. Sin embargo, la adición de SO2 a pesar de ser muy eficaz puede generar problemas de salud en los consumidores sensibles por lo que las bodegas están muy interesadas en su reducción o eliminación. Por otro lado, la filtración puede afectar a las propiedades sensoriales de los vinos y no ofrece una eficacia total en la eliminación de estos microorganismos alterantes. Por ello, recientemente se ha incrementado la búsqueda de nuevas técnicas o métodos para una mejora en el control de bacterias y levaduras alterantes en el vino sin afectar a sus propiedades sensoriales. Entre ellas la tecnología de Pulsos Eléctricos de Alto Voltaje (PEAV) se ha demostrado ampliamente eficaz en la inactivación de formas vegetativas de bacterias y levaduras alterantes presentes en el vino en diferentes etapas de su proceso de elaboración.<br /

Enzymatic Processes Triggered by PEF for Astaxanthin Extraction From Xanthophyllomyces dendrorhous

The aim of this study was to evaluate the potential of pulsed electric fields (PEF) to improve the extraction of the lipid-soluble astaxanthin from fresh biomass of a wild-type (CECT 11028) and mutant (ATCC 74219) Xanthophyllomyces dendrorhous strain using ethanol as solvent. Inactivation and propidium uptake studies revealed that inactivation is a good index for estimated the proportion of irreversible permeabilized cells when inactivation is higher than 70% in the two strains. Ethanol was ineffective for extracting carotenoids from the PEF-treated cells (20 kV/cm, 135 µs) of the two strains. However, after aqueous incubation of PEF-treated X. dendrorhous ATCC 74219 cells for 12 h, up to 2.4 ± 0.05 mg/g dried weight (d.w.) of carotenoids were extracted in ethanol. From total carotenoid extracted, around 84% corresponded to all-trans astaxanthin. The detection and quantification of esterase activity in the supernatant and the relationship between the percentage of esterase activity quantified and the amount of carotenoids extracted indicate that the extraction of astaxanthin was mediated by enzymatic esterase activity triggered by PEF during incubation. On the other hand, the formation of a large lipid globule into the cytoplasm of PEF-treated X. dendrorhous CECT 11028 cells during aqueous incubation prevented carotenoid extraction. The process developed in this investigation represents a more sustainable and greener method that those previously used for extracting astaxanthin from yeast

Microbial Decontamination by Pulsed Electric Fields (PEF) in Winemaking

Pulsed Electric Fields (PEF) is a non-thermal technique that causes electroporation of cell membranes by applying very short pulses (μs) of a high-intensity electric field (kV/cm). Irreversible electroporation leads to the formation of permanent conductive channels in the cytoplasmic membrane of cells, resulting in the loss of cell viability. This effect is achieved with low energy requirements and minimal deterioration of quality. This chapter reviews the studies hitherto conducted to evaluate the potential of PEF as a technology for microbial decontamination in the winemaking process for reducing or replacing the use of SO2, for guaranteeing reproducible fermentations or for wine stabilization

Synergetic effect of combining PEF treatments with sublethal doses of SO2 on the inactivation of Saccharomyces bayanus and Brettanomyces bruxellensis in red wine

Certain microorganisms are capable of proliferating in wine despite its low pH and high ethanol content. The yeasts of the Saccharomyces genus responsible for alcoholic fermentation can alter wines with residual sugars; the proliferation of Brettanomyces bruxellensis brings about thoroughly unpleasant sensory changes. The main strategy currently applied in wineries for microbial control is the addition of sulfites (SO2). However, sulfites are being researched due to the symptoms they can cause in allergic individuals. Pulsed electric field (PEF) technology has the capability of inactivating vegetative cells of microorganisms at non-lethal temperatures and could thus prove to be an alternative to SO2. In this study, the resistance of Saccharomyces bayanus and B. bruxellensis suspended in wine to a series of different PEF treatments (10–25 kV/cm; 25–1000 µs; 40–170 kJ/kg) combined with sublethal concentrations of SO2 (10, 25, and 50 ppm) was evaluated. The results showed that even the least intense PEF treatments (10 kV/cm; 115 kJ/kg) inactivated more than 4.0 Log10 cycles in both types of yeasts immediately after treatment. The subsequent incubation of the treated yeasts for 24 h in wine managed to increase inactivation by 3.0 Log10 cycles. The combination of a moderate PEF treatment with sublethal doses of SO2 had a synergistic lethal effect on the two yeasts under study after 24 h of incubation in wine, leading to counts lying below the detection limit (&gt;5.0 Log10 cycles). This synergistic effect was attributed to the existence of a portion of the population that had been sublethally damaged by PEF and in which SO2 could more easily penetrate the cytoplasm. These results demonstrate the capacity of PEF technology for microbial control of spoilage yeasts in wine. PEF could thus represent an alternative with the potential of eliminating or reducing SO2 levels in the winemaking process