A natural butter glyceride as a plasticizer for improving thermal, mechanical, and biodegradable properties of poly(lactide acid).

Polylactic acid (PLA) is a biobased and biodegradable thermoplastic polyester with great potential to replace petroleum-based plastics. However, its poor toughness and slow biodegradation rate affect broad applications of PLA in many areas. In this study, a glycerol triester existing in natural butter, glycerol tributyrate, was creatively explored and compared with previously investigated triacetin and tributyl citrate, as potential plasticizers of PLA for achieving improved mechanical and biodegradation performances. The compatibilities of these agents with PLA were assessed quantitively via the Hansen solubility parameter (HSP) and measured by using different testing methods. The incorporation of these compounds with varied contents ranging from 1 to 30 % in PLA altered thermal, mechanical, and biodegradation properties consistently, and the relationship and impacts of chemical structures and properties of these agents were systematically investigated. The results demonstrated that glycerol tributyrate is a novel excellent plasticizer for PLA and the addition of this triester not only effectively reduced the glass transition, cold crystallization, and melting temperatures and Young's modulus, but also led to a significant improvement in the enzymatic degradation rate of the plasticized PLA. This study paves a way for the development of sustainable and eco-friendly food grade plasticized PLA products.

Effect of pH Buffer and Carbon Metabolism on the Yield and Mechanical Properties of Bacterial Cellulose Produced by Komagataeibacter hansenii ATCC 53582.

Bacterial cellulose (BC) is widely used in the food industry for products such as nata de coco. The mechanical properties of BC hydrogels, including stiffness and viscoelasticity, are determined by the hydrated fibril network. Generally, Komagataeibacter bacteria produce gluconic acids in a glucose medium, which may affect the pH, structure and mechanical properties of BC. In this work, the effect of pH buffer on the yields of Komagataeibacter hansenii strain ATCC 53582 was studied. The bacterium in a phosphate and phthalate buffer with low ionic strength produced a good BC yield (5.16 and 4.63 g/l respectively), but there was a substantial reduction in pH due to the accumulation of gluconic acid. However, the addition of gluconic acid enhanced the polymer density and mechanical properties of BC hydrogels. The effect was similar to that of the bacteria using glycerol in another carbon metabolism circuit, which provided good pH stability and a higher conversion rate of carbon. This study may broaden the understanding of how carbon sources affect BC biosynthesis.

Contribution of the mitogen-activated protein kinase Hog1 to the halotolerance of the marine yeast Debaryomyces hansenii.

Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1 M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.

Effects of initial oxygenation on chemical and aromatic composition of wine in mixed starters of Hanseniaspora vineae and Saccharomyces cerevisiae.

The use of Saccharomyces and non-Saccharomyces yeast species as mixed starters has potential advantages over pure culture fermentation due to increased wine complexity based on modification of metabolites of oenological interest. In this work, the effects of initial oxygenation on fermentation performance, chemical and volatile composition of French Colombard wine fermented with Hanseniaspora vineae and Saccharomyces cerevisiae in sequential inoculations were investigated in 1 L flasks. Although dominated by S. cerevisiae at the middle-end of fermentation, initial aeration for 1 day boosted H. vineae populations, and allowed H. vineae to coexist longer with S. cerevisiae in mixed cultures compared to no aeration, and suppressed S. cerevisiae later in the fermentation, which resulted in extended fermentation time. More important, the major fermentation products and volatile compounds were significantly modified by aeration and different from no aeration fermentation. The wines produced by aeration of mixed fermentations were characterized with higher amounts of glycerol, lactic acid and acetate esters, and lower levels of ethanol, higher alcohol and ethyl fatty acid esters. The aeration had more potential to shape the quality of wines and diversify the aromatic characteristics relative to simple mixed inoculation, as indicated by PCA analysis. Our results suggested that the impact of early aeration on yeast physiology extends beyond the aeration phase and influences fermentation activity, chemical and aromatic compounds in the following anaerobic stage. The aeration for a short time during the cell growth stage in mixed fermentation is therefore a potential means to increase the aromatic diversity and quality of wine, possibly providing an alternative approach to meet the expectations of wine consumers for diverse aromatic qualities.

Green extraction of polyphenols from grapefruit peels using high voltage electrical discharges, deep eutectic solvents and aqueous glycerol.

Deep eutectic solvents (DES) and aqueous glycerol were proposed as green alternatives to conventional solvents for the extraction of polyphenols from grapefruit peels. In order to increase the extraction kinetics and yields of polyphenols, high voltage electrical discharges (HVED) were used as a pre-treatment technology (energy varied between 7.27 and 218 kJ/kg). Results showed that the HVED energy input can be reduced, when the subsequent solid-liquid extraction was performed in 20% (w/v) aqueous glycerol or in DES (lactic acid: glucose) instead of water. The addition of glycerol has reduced the energy of the pre-treatment by 6 times. The same diffusivity of polyphenols (4 × 10-11 m2/s) was obtained in water from HVED pre-treated peels at 218 kJ/kg and in aqueous glycerol from pre-treated peels at 36 kJ/kg. The solubility of naringin, the main flavonoid compound of grapefruit peels in the solvents, was investigated through a theoretical modelling of its Hansen solubility parameters.

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.

Vacuolar control of subcellular cation distribution is a key parameter in the adaptation of Debaryomyces hansenii to high salt concentrations.

Debaryomyces hansenii is a halotolerant and Na+-includer yeast that can be isolated from different food and low-water activity products. It has also been defined as a marine-occurring yeast but key aspects for this salt tolerant behavior are far from being understood. Here, we searched for clues helping to elucidate the basis of this ability. Our results on growth, Rb+ transport, total K+ and Na+ content and vacuolar fragmentation are compatible with a yeast species adapted to cope with salt stress. On the other hand, we confirmed the existence of D. hansenii strategies that are generally observed in sensitive organisms, such as the production of glycerol as a compatible solute and the efficient vacuolar sequestration of Na+. We propose a striking role of D. hansenii vacuoles in the maintenance of constant cytosolic K+ values, even in the presence of extracellular Na+ concentration values more than two orders of magnitude higher than extracellular K+. Finally, the ability to deal with cytosolic Na+ levels significantly higher than those found in S. cerevisiae, shows the existence of important and specific salt tolerance mechanisms and determinants in D. hansenii.

Characterization of new polyol/H+ symporters in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast that produces and assimilates a wide variety of polyols. In this work we evaluate polyol transport in D. hansenii CBS 767, detecting the occurrence of polyol/H(+) (and sugar/H(+)) symporter activity, through the transient extracellular alkalinization of unbuffered starved cell suspensions. From the D. hansenii genome database, we selected nine ORFs encoding putative transporter proteins to clone in a centromeric plasmid with C-terminal GFP tagging and screened for polyol/H(+) symporters by heterologous expression in Saccharomyces cerevisiae. Five distinct D. hansenii polyol/H(+) symporters were identified and characterized, with different specificities and affinities for polyols, namely one glycerol-specific (DhStl1), one D-galactitol-specific (DhSgl1, Symporter galactitol/H(+) 1), one D-(+)-chiro-inositol-specific (DhSyi1, Symporter D-(+)-chiro-inositol/H(+) 1), one for D-sorbitol/D-mannitol/ribitol/D-arabitol/D-galactitol (DhSyl1, Symporter Polyols 1) and another for D-sorbitol/D-mannitol/ribitol/D-arabitol (DhSyl2, Symporter Polyols 2). This work contributed to the annotation of new yeast polyol transporters, including two specific for uncommon substrates as galactitol and D-(+)-chiro-inositol.

An optimized procedure for the enological selection of non-Saccharomyces starter cultures.

The apiculate yeasts are the species predominating the first stage of grape must alcoholic fermentation and are important for the production of desired volatile compounds. The aim of the present investigation was to establish a protocol for the enological selection of non-Saccharomyces strains directly isolated from a natural must fermentation during the tumultuous phase. At this scope, fifty Hanseniaspora uvarum isolates were characterized at strain level by employing a new combined PCR-based approach. One isolate representative of each identified strain was used in fermentation assays to assess strain-specific enological properties. The chemical analysis indicated that all the analyzed strains were low producers of acetic acid and hydrogen sulphide, whereas they showed fructophilic character and high glycerol production. Analysis of volatile compounds indicated that one strain could positively affect, during the alcoholic fermentation process, the taste and flavour of alcoholic beverages. The statistical evaluation of obtained results indicated that the selected autochthonous H. uvarum strain possessed physiological and technological properties which satisfy the criteria indicated for non-Saccharomyces wine yeasts selection. Our data suggest that the described protocol could be advantageously applied for the selection of non-Saccharomyces strains suitable for the formulation of mixed or sequential starters together with Saccharomyces cerevisiae.

Production of arabitol from glycerol: strain screening and study of factors affecting production yield.

Glycerol is a major by-product from biodiesel production, and developing new uses for glycerol is imperative to overall economics and sustainability of the biodiesel industry. With the aim of producing xylitol and/or arabitol as the value-added products from glycerol, 214 yeast strains, many osmotolerant, were first screened in this study. No strains were found to produce large amounts of xylitol as the dominant metabolite. Some produced polyol mixtures that might present difficulties to downstream separation and purification. Several Debaryomyces hansenii strains produced arabitol as the predominant metabolite with high yields, and D. hansenii strain SBP-1 (NRRL Y-7483) was chosen for further study on the effects of several growth conditions. The optimal temperature was found to be 30°C. Very low dissolved oxygen concentrations or anaerobic conditions inhibited polyol yields. Arabitol yield improved with increasing initial glycerol concentrations, reaching approximately 50% (w/w) with 150 g/L initial glycerol. However, the osmotic stress created by high salt concentrations (≥50 g/L) negatively affected arabitol production. Addition of glucose and xylose improved arabitol production while addition of sorbitol reduced production. Results from this work show that arabitol is a promising value-added product from glycerol using D. hansenii SBP-1 as the producing strain.

Molecular and physiological characteristics of a grape yeast strain containing atypical genetic material.

The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from "Primitivo" grape (http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO2 and temperature) useful for industrial application.

Extracellular lipolytic enzyme activity of a newly isolated Debaryomyces hansenii.

A strain isolated from waste of a milk products plant and exhibited extracellular lipolytic activity was identified as Debaryomyces hansenii by 5.8S rRNA and 28S rRNA gene sequence analyses. Lipolytic activity was assayed spectrophotometrically by using p-nitrophenylpalmitate. Higher specific lipolytic activities were obtained in the presence of tristearin (0.68 U/mg prot), oleic acid (0.56 U/mg prot), and soybean oil (0.36 U/mg prot) than other triglycerides, fatty acids, and vegetable oils considered as carbon sources. Cheese whey appeared to be a good alternative to lipidic substances for lipolytic activity. Among various organic and inorganic nitrogen sources, soy flour was found to attain the lipolytic activity similar to that provided by universal yeast medium components. This work is the first report on the discussion of lipolytic activity enhancement by D. hansenii through modulating the cultivation medium. It also proposes low cost medium nutrients that could be of industrial value and could serve as basal nutrients for further optimization studies on the lipase production by D. hansenii.

Cell wall involvement in the glycerol response to high osmolarity in the halotolerant yeast Debaryomyces hansenii.

Osmotic stress was studied through the induction of the gene coding for glycerol 3-phosphate dehydrogenase (DhGPD1) in the halotolerant yeast Debaryomyces hansenii. This yeast responded to modifications in turgor pressure by stimulating the transcription of DhGPD1 when exposed to solutes that cause turgor stress (NaCl or sorbitol), but did not respond to water stress mediated by ethanol. In contrast to what has been documented to occur in Saccharomyces cerevisiae, D. hansenii protoplasts did not show induction in the transcription of DhGPD1 showing a limitation in their response to solute stress. The results presented indicate that the presence of the cell wall is of significance for the induction of DhGPD1 and hence for osmotic regulation in halotolerant D. hansenii. It appears that the main osmosensor that links high osmolarity with glycerol accumulation may be of a different nature in this yeast.

Expression of the GPD1 and GPP2 orthologues and glycerol retention during growth of Debaryomyces hansenii at high NaCl concentrations.

The highly NaCl-tolerant yeast Debaryomyces hansenii produces and obtains high levels of intracellular glycerol as a compatible solute when grown at high NaCl concentrations. The effect of high NaCl concentrations (4%, 8% and 12% w/v) on the glycerol production and the levels of intra- and extracellular glycerol was determined for two D. hansenii strains with different NaCl tolerance and compared to one strain of the moderately NaCl-tolerant yeast Saccharomyces cerevisiae. Initially, high NaCl tolerance seems to be determined by enhanced glycerol production, due to an increased expression of DhGPD1 and DhGPP2 (AL436338) in D. hansenii and GPD1 and GPP2 in S. cerevisiae; however, the ability to obtain high levels of intracellular glycerol seems to be more important. The two D. hansenii strains had higher levels of intracellular glycerol than the S. cerevisiae strain and were able to obtain high levels of intracellular glycerol, even at very high NaCl concentrations, indicating the presence of, for example, a type of closing channel, as previously described for other yeast species.

Heterologous expression of glycerol 3-phosphate dehydrogenase gene [DhGPD1] from the osmotolerant yeast Debaryomyces hansenii in Saccharomyces cerevisiae.

The role for the gene encoding glycerol 3-phosphate dehydrogenase (DhGPD1) from the osmotolerant yeast Debaryomyces hansenii, in glycerol production and halotolerance, was studied through its heterologous expression in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Delta). The expression of the DhGPD1 gene in the gpd1Delta background restored glycerol production and halotolerance to wild type levels, corroborating its role in the salt-induced production of glycerol. Although the gene was functional in S. cerevisiae, its heterologous expression was not efficient, suggesting that the regulatory mechanism may not be shared by these two yeasts.

Mechanisms underlying the halotolerant way of Debaryomyces hansenii.

The yeast Debaryomyces hansenii is usually found in salty environments such as the sea and salted food. It is capable of accumulating sodium without being intoxicated even when potassium is present at low concentration in the environment. In addition, sodium improves growth and protects D. hansenii in the presence of additional stress factors such as high temperature and extreme pH. An array of advantageous factors, as compared with Saccharomyces cerevisiae, is putatively involved in the increased halotolerance of D. hansenii: glycerol, the main compatible solute, is kept inside the cell by an active glycerol-Na+ symporter; potassium uptake is not inhibited by sodium; sodium protein targets in D. hansenii seem to be more resistant. The whole genome of D. hansenii has been sequenced and is now available at http://cbi.labri.fr/Genolevures/ and, so far, no genes specifically responsible for the halotolerant behaviour of D. hansenii have been found.

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress.

The comparative analysis of growth, intracellular content of Na+ and K+, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na+ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.

Physiological behaviour of Hanseniaspora guilliermondii in aerobic glucose-limited continuous cultures.

The physiology of Hanseniaspora guilliermondii was studied under aerobic glucose-limited conditions using the accelerostat procedure (continuous acceleration of dilution rate) and classical chemostat cultures. By both cultivation techniques this yeast was found to be Crabtree-positive. Up to a dilution rate of 0.25 h(-1), glucose was completely metabolised into biomass, glycerol and carbon dioxide. Above this value, an increase in the dilution rate was accompanied by the production of other metabolites like ethanol, acetic and malic acids. Biomass yield during the purely oxidative growth was 0.49 g g(-1) and decreased to 0.26 g g(-1) for D=0.34 h(-1). A maximal specific ethanol production rate of 1.36 mmol g(-1) h(-1) and a maximal ethanol yield of 0.05 g g(-1) were achieved at D=0.34 h(-1).

Active glycerol uptake is a mechanism underlying halotolerance in yeasts: a study of 42 species.

A comparison of 42 yeast species with respect to growth in the presence of high NaCl concentration and characteristics of glycerol uptake is presented. The yeast species were classified into four classes on the basis of their ability to grow in the presence of 1, 2, 3 or 4 M NaCl. Considering that two different types of active-transport systems for glycerol uptake have been described, Na+/glycerol and H+/glycerol symports, glycerol transport was investigated by testing for proton uptake upon glycerol addition in cells incubated in the absence and in the presence of NaCl. Only strains belonging to the two higher classes of salt tolerance showed constitutive active glycerol uptake, and could accumulate glycerol internally against a concentration gradient. Five of these strains exhibited a H+/glycerol symport. All the other strains showed evidence of the activity of a salt-dependent glycerol uptake similar to that described in the literature for Debraryomyces hansenii. The strains within the two lower classes of salt tolerance showed, to varying degrees, glycerol active uptake only when glycerol was used as the carbon and energy source, suggesting that this uptake system is involved in glycerol catabolism. The results within this work suggest that active glycerol uptake provides a basis for high halotolerance, helping to maintain a favourable intracellular concentration of glycerol. The relation between the constitutive expression of such carriers and a higher level of salt-stress resistance suggests that this may be an evolutionary advantage for growth under such conditions.

Glycerol and other fermentation products of apiculate wine yeasts.

Ninety-six strains of apiculate wine yeasts were studied for their ability to produce glycerol, acetaldehyde, ethyl acetate, sulphur dioxide and hydrogen sulphide in synthetic medium. Hanseniaspora guilliermondii produced smaller quantities of glycerol, acetaldehyde and hydrogen sulphide than Kloeckera apiculata, whereas the production of ethyl acetate and sulphur dioxide was found to be similar. Strains characterized by different capacities and properties were found for both species. The existence of apiculate strains differing in secondary compound production is of technological interest, as these yeasts constitute potential flavour producers. Selected strains of apiculate yeasts might favour an enhanced flavour formation and yield desirable characteristics to the final product.