Dehydration stress responses of yeasts Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans: Effects of glutathione and trehalose biosynthesis.

In food industry and winemaking, the use of active dehydrated yeast (ADY) Saccharomyces cerevisiae is a frequent practice because of the long-term stability and high efficiency of ADY. Nowadays, there is an increasing interest for new yeasts strains, such as Torulaspora delbrueckii (Td), Metschnikowia pulcherrima (Mp) and Lachancea thermotolerans (Lt). However, the yeasts transformation processes into the solidified form generate several stresses that reduce the cell viability. In this case, understanding the phenomena of yeast cell resistance before, during and after dehydration is of great importance. In this study we analyzed two compounds associated with resistance to stress and produced by cells, glutathione (total, oxidized and reduced) and trehalose, at different stages of the process. The impact of growing and dehydration conditions on cell viability was analyzed by flow cytometry and two-photon laser scanning microscopy. The results showed that cells naturally enriched in glutathione or trehalose acquired resistance to dehydration, preventing the oxidation of glutathione in a growth/dehydration condition dependent manner. This is the first time that simultaneous metabolic and dehydration responses were observed in three non-Saccharomyces strains. These findings represent an opportunity to better understand the yeast's dehydration resistance phenomena and thus to promote the efficient industrial production of new dried yeasts.

Selected dehydrogenases in Yarrowia lipolytica JMY 861: their role in the synthesis of flavor compounds.

The presence of selected dehydrogenases, including alcohol dehydrogenase (ADH-YL) and aldehyde dehydrogenase (ALDH-YL), in Yarrowia lipolytica JMY 861, and their potential role in flavor synthesis were investigated. The experimental findings showed that using reduced form of nicotinamide adenine dinucleotide (NADH) as cofactor, the ADH-YL activity in vitro was 6-fold higher than that with reduced form of nicotinamide adenine dinucleotide phosphate (NADPH); however, under the experimental conditions used in this study, an ALDH-YL activity was not detected. The in situ hexanal reduction reaction was found to be instantaneous; however, when the yeast cells suspension was diluted 150 times, the initial relative hexanal concentration was increased by 84.1%. The chromatographic analyses indicated the conversion, in situ, of linoleic acid hydroperoxides (HPODs) into volatile C6-compounds after 60 min of HPODs addition to the yeast cells suspension.

For me the taste of soy is not a barrier to its consumption. And how about you?

This research investigates the cultural influence on beliefs about and attitudes towards soy foods (French from France vs. Vietnamese from Vietnam) and possible change of beliefs and attitudes and soy consumption habits with a change in cultural environment (French from France vs. French from Vietnam, Vietnamese from Vietnam vs. Vietnamese from France) of French and Vietnamese participants, two countries with very different soy food consumption. Expressed beliefs and attitudes of soy foods resulting from discussions in focus groups, conducted in both countries, were collected and used to derive a questionnaire. French participants differ mainly from Vietnamese participants in questions associated to taste and price of soy foods. Both groups reported positive attitudes towards health benefits of soy foods. With a change in cultural environment, French participants showed a notable change in attitudes related to taste and price of soy foods and in soy consumption habit whereas almost no change was observed in Vietnamese participants. The asymmetry in magnitude of change and cultural differences in components of beliefs and attitudes are discussed.

A novel method to assess heat transfer and impact of relevant physicochemical parameters for the scaling up of solid state fermentation systems.

Heat production during fermentation is undesirable. It can affect the growth of biomass, sporulation, products formation and the scaling-up. Physico-chemical properties of substrates influence heat and mass transfer in Solid State Fermentation. Heat is chemically produced into substrates without micro-organism to allow better reproducibility. A 24-1 fractional factorial design is chosen to study the influence of four physicochemical parameters on heat transfer: Granulometry, Bulk Density, Carr Index (compressibility index) and Water Absorption Capacity. Results show that the two main physicochemical parameters which influence heat transfer are Granulometry and Carr Index. High Granulometry and low Carr Index have influence on maximum temperature reached during the test, warm-up speed and cooling speed. These two parameters allow efficient air flow through the substrate bed with large interparticle spaces enhancing exchange surface between air and particles. A substrate with these characteristics facilitates heat transfers in forced-aerated reactors.

Highlighting Protective Effect of Encapsulation on Yeast Cell Response to Dehydration Using Synchrotron Infrared Microspectroscopy at the Single-Cell Level.

In the present paper, the Layer by Layer (LbL) method using ß-lactoglobulin and sodium alginate was performed to individually encapsulate Saccharomyces cerevisiae cells in microorganized shells in order to protect them against stresses during dehydration. Higher survival (∼1 log) for encapsulated yeast cells was effectively observed after air dehydration at 45°C. For the first time, the potentiality of Synchrotron-Fourier Transform InfraRed microspectroscopy (S-FTIR) was used at the single-cell level in order to analyze the contribution of the biochemical composition of non-encapsulated vs. encapsulated cells in response to dehydration. The microspectroscopy measurements clearly differentiated between non-encapsulated and encapsulated yeast cells in the amide band region. In the spectral region specific to lipids, the S-FTIR results indicated probably the decrease in membrane fluidity of yeast after dehydration without significant distinction between the two samples. These data suggested minor apparent chemical changes in cell attributable to the LbL system upon dehydration. More insights are expected regarding the lower mortality among encapsulated cells. Indeed the hypothesis that the biopolymeric layers could induce less damage in cell by affecting the transfer kinetics during dehydration-rehydration cycle, should be verified in further work.

Biophysical Stress Responses of the Yeast Lachancea thermotolerans During Dehydration Using Synchrotron-FTIR Microspectroscopy.

During industrial yeast production, cells are often subjected to deleterious hydric variations during dehydration, which reduces their viability and cellular activity. This study is focused on the yeast Lachancea thermotolerans, particularly sensitive to dehydration. The aim was to understand the modifications of single-cells biophysical profiles during different dehydration conditions. Infrared spectra of individual cells were acquired before and after dehydration kinetics using synchrotron radiation-based Fourier-transform infrared (S-FTIR) microspectroscopy. The cells were previously stained with fluorescent probes in order to measure only viable and active cells prior to dehydration. In parallel, cell viability was determined using flow cytometry under identical conditions. The S-FTIR analysis indicated that cells with the lowest viability showed signs of membrane rigidification and modifications in the amide I (α-helix and ß-sheet) and amide II, which are indicators of secondary protein structure conformation and degradation or disorder. Shift of symmetric C-H stretching vibration of the CH2 group upon a higher wavenumber correlated with better cell viability, suggesting a role of plasma membrane fluidity. This was the first time that the biophysical responses of L. thermotolerans single-cells to dehydration were explored with S-FTIR. These findings are important for clarifying the mechanisms of microbial resistance to stress in order to improve the viability of sensitive yeasts during dehydration.

Hemp (Cannabis sativa L.) Protein Extraction Conditions Affect Extraction Yield and Protein Quality.

The purpose of this paper was to study the extraction conditions of hemp proteins from undelipidated press-cakes. The effects of different hydration conditions on protein recovery yield and polypeptide profile were evaluated: pH (2 to 12), ionic strength (0 to 500 mM NaCl) and press-cake/liquid weight ratio (2% to 22%). pH was the most impacting factor. At acidic pH (2 to 7) the extraction yields were low and quite constant (<7%), corresponding mainly to hemp albumins solubilization. The extraction of globulins started to increase significantly from pH 8, with protein extraction yield varying from 8.3% at pH 8 to 67.1% at pH 12 for a 10% press-cake/liquid weight ratio. The addition of NaCl in press-cake suspensions did not increase the total nitrogen content in aqueous extracts at alkaline pH while the lowest press-cake/liquid weight ratios (5% to 10%) were revealed optimal regarding protein recovery rate. The intense coloration observed on the aqueous extracts above pH 8 was assigned to solubilization and oxidation of phenolic compounds whose concentration increased about sevenfolds from pH 2 to 12. At the highest applied pH (11 to 12), the formation of covalent complexes between phenolic compounds and some hemp polypeptides was hypothesized. Aqueous extraction at strong alkaline pH (>9) without salt addition and respecting a 10% press-cake/liquid weight ratio should be retained to optimize protein extraction yield. However, further purification steps are required to evaluate the nutritional, organoleptic, and techno-functional properties of hemp proteins extracted in such conditions. PRACTICAL APPLICATION: The traditional extraction process of hemp proteins by alkaline solubilization and isoelectric precipitation, mostly from delipidated hemp press-cake, leads to limited quantity and poor solubility of extracted proteins, and data related to extraction conditions are insufficiently available to optimize this process. This article aims to find optimal hydration conditions (pH, ionic strength, press-cake to liquid ratio) for protein extraction from undelipidated hemp press-cake, allowing high protein recovery and preserving protein quality. The results obtained represent very useful data for developing an economically viable and sustainable extraction process of proteins from raw hemp press-cake.

Oxidative stress resistance during dehydration of three non-Saccharomyces wine yeast strains.

The development of new fermented foods and beverages requires more and more the use of new dehydrated yeasts species. In this context, the non-Saccharomyces (NS) yeasts Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans are developed especially in winemaking as co-culture in the fermentation of the must or for the must bioprotection. However, during formulation-dehydration the yeast cells are exposed to several stresses that reduce cellular activity. Among these, the oxidative stress induced by the stabilization processes has been described as one of the main causes of cell death. In this study, we analyzed the effects of growth medium associated with two dehydration kinetics on the accumulation of reactive oxygen species (ROS) and lipid peroxidation levels. The cultivability of tested yeast strains was dependent on growth and dehydration conditions. The L. thermotolerans strain was the most sensible to dehydration when growing in nutrient-poor media, which was characterized by high levels of ROS, lipid peroxidation and reduced cultivability. In contrast, this yeast was able to restore its cultivability when growing in nutrient-rich medium before dehydration. The other NS yeast strains acquired resistance to oxidative stress similarly but in a growth-dehydration condition less dependent manner. These results showed that modulation of growing medium composition is a simple way to improve resistance to oxidative attack imposed by dehydration in NS yeasts. This was the first time that multiple quantitative and qualitative indicators of oxidative stress response in these three NS yeast strains were explored.

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: implication of their membrane fluidizing action.

Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called "green notes". The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of HPOD on yeast membrane. In addition, we determined by infrared spectroscopy measurement that this compound rapidly diffused into model phospholipids [1, 2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-D54)] bilayer, modifying their general physical state and their phase transition. In the presence of various concentrations of HPOD, the phase transition of DMPC-D54 occurred with an increase of both the corresponding wave number shift and the temperature range but the phase transition temperature was not modified. These results show that the toxic effects of HPOD on the yeast Yarrowia lipolytica may be initially linked to a strong interaction of this compound with the cell membrane phospholipids and components.

Size measuring techniques as tool to monitor pea proteins intramolecular crosslinking by transglutaminase treatment.

In this work, techniques for monitoring the intramolecular transglutaminase cross-links of pea proteins, based on protein size determination, were developed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of transglutaminase-treated low concentration (0.01% w/w) pea albumin samples, compared to the untreated one (control), showed a higher electrophoretic migration of the major albumin fraction band (26 kDa), reflecting a decrease in protein size. This protein size decrease was confirmed, after DEAE column purification, by dynamic light scattering (DLS) where the hydrodynamic radius of treated samples appears to be reduced compared to the control one.

Aqueous two-phase system cold-set gelation using natural and recombinant probiotic lactic acid bacteria as a gelling agent.

The present study aimed to entrap probiotic lactic acid bacteria (LAB) in a sodium alginate and sodium caseinate aqueous two-phase gel system. The natural acidifying properties of two therapeutic probiotic LAB were exploited to liberate calcium ions progressively from calcium carbonate (CaCO3), which caused the gelation of the co-existing phases. Bi-biopolymeric matrix gelation of GDL/CaCO3 or LAB/CaCO3 was monitored by dynamic rheological measurements, and the final gels were characterized by frequency dependence measurements and confocal laser scanning microscopy. Weak to strong gels were formed with an elastic modulus G' from 10 to 1.000Pa, respectively. After cold-set gelation of our system, confocal laser scanning microscopy showed spherical protein microdomains trapped within a calcium alginate network. LAB cells were stained to study their partition in the self-gelling matrices. Our LAB strains showed two different behaviors, which may relate to the exopolysaccharide production: (i) Lactobacillus plantarum CNRZ1997 cells were found mainly in continuous alginate networks, whereas (ii) Lactococcus lactis cells were localized in protein microdomains. This alginate-caseinate phase-separated system that was self-gelled by LAB cells may be an innovative approach for immobilizing and protecting LAB cells.

Optimization of the Hydrolysis of Safflower Oil for the Production of Linoleic Acid, Used as Flavor Precursor.

Commercial lipases, from porcine pancreas (PPL), Candida rugosa (CRL), and Thermomyces lanuginosus (Lipozyme TL IM), were investigated in terms of their efficiency for the hydrolysis of safflower oil (SO) for the liberation of free linoleic acid (LA), used as a flavor precursor. Although PPL, under the optimized conditions, showed a high degree of hydrolysis (91.6%), its low tolerance towards higher substrate concentrations could limit its use for SO hydrolysis. In comparison to the other investigated lipases, Lipozyme TL IM required higher amount of enzyme and an additional 3 h of reaction time to achieve its maximum degree of SO hydrolysis (90.2%). On the basis of the experimental findings, CRL was selected as the most appropriate biocatalyst, with 84.1% degree of hydrolysis. The chromatographic analyses showed that the CRL-hydrolyzed SO is composed mainly of free LA.

A new alternative in vitro method for quantification of Toxoplasma gondii infectivity.

An in vitro method to determine the infectious potency of an unknown suspension of the protozoan parasite Toxoplasma gondii based on kinetics of host cells lysis was developed. Mic1-3KO a mutant strain of T. gondii RH tachyzoites was inoculated in 25-cm² flasks containing a 90% confluent monolayer of human foreskin fibroblasts. Lysis kinetics was monitored for infection ratios ranging from 1∶106 to 1∶10; we defined 106 tachyzoites/ml⁻¹ as the threshold value for parasite egress. Results allowed us to build a calibration curve relating the initial infection ratios to the time needed to reach 106 tachyzoites/ml⁻¹. Finally, we validated the method using a known mixture of dead and live parasites. This method was found to estimate with accuracy the initial ratio of infection of the unknown parasite suspension. This easy-to-use method is reproducible and can be applied to any T. gondii tachyzoite RH strain, genetically modified or not. This method is also suitable for testing promising candidates for an effective live vaccine.

Comparison of some new pretreatment methods for second generation bioethanol production from wheat straw and water hyacinth.

Pretreatment of lignocellulosic residues like water hyacinth (WH) and wheat straw (WS) using crude glycerol (CG) and ionic liquids (IL) pretreatment was evaluated and compared with conventional dilute acid pretreatment (DAT) in terms of enzymatic hydrolysis yield and fermentation yield of pretreated samples. In the case of WS, 1-butyl-3-methylimidazolium acetate pretreatment was found to be the best method. The hydrolysis yields of glucose and total reducing sugars were 2.1 and 3.3 times respectively higher by IL pretreatment than DAT, while it was 1.4 and 1.9 times respectively higher with CG pretreatment. For WH sample, CG pretreatment was as effective as DAT and more effective than IL pretreatment regarding hydrolysis yield. The fermentation inhibition was not noticeable with both types of pretreatment methods and feedstocks. Besides, CG pretreatment was found as effective as pure glycerol pretreatment for both feedstocks. This opens up an attractive economic route for the utilization of CG.

Metabolism of phenylalanine and biosynthesis of styrene in Penicillium camemberti.

The occurrence of styrene in food may be an important aroma defect (celluloid odour), even at very low concentrations (Miltz et al. 1980) causing consumer rejection and is therefore a problem for the food industry. We examined the biosynthetic pathway leading to styrene formation by Penicillium camemberti using labelled compounds. As styrene is strongly hydrophobic and volatile, we first had to develop a continuous extraction process. Using resins XAD2 it was reasonable to suspect phenylalanine (Phe) as the precursor. The addition of Phe marked with 13C on the ring provokes the accumulation of labelled styrene. The enzyme activities involved were also tentatively measured. Styrene appears to be synthesized from phenylalanine by phenylalanine ammonia-lyase activity followed by a decarboxylation catalysed by a cinnamic acid decarboxylase.