Resveratrol in Parts of Vine and Wine Originating from Bohemian and Moravian Vineyard Regions

Chemically, resveratrol is a substance of a polyphenolic character from the group of phytoalexins - 3,5,4´- trihydroxystilbene - and exists in cis and trans-isomer forms. In natural sources trans-isomer is more common. As a natural polyphenolic substance, it shows a whole range of biological activities, such as anti-oxidizing and anti-microbial features (namely anti-fungal activities), the ability to absorb free radicals, affects blood sedimentation rate etc. Recently, trans-resveratrol has also been attributed anti-mutagen and chemo-protective features against cancer proliferation. It is assumed that resveratrol could be one of the active substances contributing to the health benefits, namely it decreases the risk of cardiovascular diseases through a reasonable consumption of red wine. Grapes of Vitis vinifera and especially red wine represent its main source in human diet. Grape peels contain about 0.5 to 2.0 mg of resveratrol/g of dry weight and the average concentration in red wines of world provenience fluctuates between 1.0 and 3.0 mg/l. Resveratrol was determined by HPLC method with electrochemical detection after direct injection of wine or plant extracts. As expected, red wines from vines originating in the Bohemian and Moravian vineyard regions appeared to contain relatively high levels of resveratrol (from 1.3 to 15.4 mg/l) and trans/cis ratio ranged from 0.5 to 4.8, excess of cis-resveratrol to trans-isomer was typical for red wine growing in Most region (northern Bohemia) where vineyards are exposed to higher environmental stress due to frequent air pollutions in this area. In addition, resveratrol determined in different parts of grapevine (leaves, rachis) varied from 6 to 490 mg/kg of the dry matter. Cluster stems were found as the richest source of resveratrol

Effects of glycerol supply and specific growth rate on methanol-free production of CALB by P. pastoris : functional characterisation of a novel promoter

As Pichia pastoris (syn. Komagataella sp.) yeast can secrete pure recombinant proteins at high rates, it is a desirable production system. The function of a novel synthetic variant of the AOX1 promoter was characterised comprehensively using a strain secreting Candida antarctica lipase B (CALB) as a model. A new time-saving approach was introduced to determine, in only one experiment, the hitherto unknown relationship between specific product formation rate (qp) and specific growth rate (μ). Tight control of recombinant protein formation was possible in the absence of methanol, while using glycerol as a sole carbon/energy source. CALB was not synthesised during batch cultivation in excess glycerol (>10 g l-1) and at a growth rate close to μmax (0.15 h−1). Between 0.017 and 0.115 h−1 in glycerol-limited fedbatch cultures, basal levels of qp > 0.4 mg g−1 h−1 CALB were reached, independent of the μ at which the culture grew. At μ > 0.04 h−1, an elevated qp occurred temporarily during the first 20 h after changing to fedbatch mode and decreased thereafter to basal. In order to accelerate the determination of the qp(μ) relationship (kinetics of product formation), the entire μ range was covered in a single fedbatch experiment. By linearly increasing and decreasing glycerol addition rates, μ values were repeatedly shifted from 0.004 to 0.074 h−1 and vice versa. Changes in qp were related to changes in μ. A rough estimation of μ range suitable for production was possible in a single fedbatch, thus significantly reducing the experimental input over previous approaches comprising several experiments

Study of the fermentative activity of Hansenula anomala and production of chemical compounds of sensory importance

Se ha estudiado la actividad fermentativa de Hansenula anomala RIVE 7-1-5 con el objetivo de evaluar la producción de compuestos químicos de importancia sensorial. Los resultados mostraron que fermenta bien monosacáridos y también sucrosa y maltosa. Su actividad fermentativa es inhibida a concentraciones de 100,0mg/L de metabisulfito de sodio en el medio. Además, es capaz de producir 5,81±0,1 % v/v de etanol. La agitación del medio de cultivo incrementa la producción de alcoholes superiores (679,2 mg/L) y etil acetato (206,0±8,0 mg/L), por el contrario disminuye la producción de ácido acético (196,0±7,0 mg/L). La producción de glicerol fue similiar tanto en cultivo estático (sin agitación) como agitado. Durante el cultivo batch en biorreactor a condiciones aireadas la tasa de crecimiento (μ) alcanzó el valor de 0,13 h-1 y se observó la producción de ácido acético hasta 4,2±0,3 g/L. La concentración de oxígeno en el medio afecta su metabolismo, así cantidades insuficientes de oxígeno provocaría un metabolismo respirofermentativo con la producción de etanol, alcoholes superiores, ésteres y ácido acético. El control de la aireación al medio de fermentación es una herramienta útil para controlar el balance entre la actividad respiratoria y fermentativa y así la síntesis de compuestos de importancia sensorial en la producción de bebidas fermentadas no tradicionales.The fermentative behaviour of Hansenula anomala RIVE 7-1-5 was studied in order to evaluate the production of chemical compounds of sensory importance. The results demonstrated that the strain ferments very well monosaccharides and also sucrose and maltose. Its fermentative activity was inhibited at concentrations of 100 mg/L of sodium metabisulphite in the medium. Furthermore, it was able to produce 5,81±0,1% (v/v) of ethanol. Agitation of the culture medium increases the production of higher alcohols (679,2 mg/L) and ethyl acetate (206,0±8,0 mg/L), but on the contrary affects the production of acetic acid (196,0±7,0mg/L). Glycerol production was similar in static (without agitation) and shaken cultivation. During batch cultivation carried out in biorreactor under aerated conditions the growth rate (μ) reached value of 0,13 h-1 and, it was also observed production of acetic acid at levels of 4,2±0,3 g/L. The oxygen concentration in the medium affects its metabolism, thus insufficient amounts of oxygen would provoke a respirofermentative metabolism with production of ethanol, higher alcohols, esters and acetic acid. The control of aeration during fermentation is a useful tool to control the balance between the respiratory and fermentative activity and thus; synthesis of compounds of sensory importance in the production of non-traditional fermented beverages

Single-Cell Approach to Monitor the Unfolded Protein Response During Biotechnological Processes With Pichia pastoris

Pichia pastoris (Komagataella sp.) is broadly used for the production of secreted recombinant proteins. Due to the high rate of protein production, incorrectly folded proteins may accumulate in the endoplasmic reticulum (ER). To restore their proper folding, the cell triggers the unfolded protein response (UPR); however, if the proteins cannot be repaired, they are degraded, which impairs process productivity. Moreover, a non-producing/non-secreting subpopulation of cells might occur, which also decreases overall productivity. Therefore, an in depth understanding of intracellular protein fluxes and population heterogeneity is needed to improve productivity. Under industrially relevant cultivation conditions in bioreactors, we cultured P. pastoris strains producing three different recombinant proteins: penicillin G acylase from Escherichia coli (EcPGA), lipase B from Candida antarctica (CaLB) and xylanase A from Thermomyces lanuginosus (TlXynA). Extracellular and intracellular product concentrations were determined, along with flow cytometry-based single-cell measurements of cell viability and the up-regulation of UPR. The cell population was distributed into four clusters, two of which were viable cells with no UPR up-regulation, differing in cell size and complexity. The other two clusters were cells with impaired viability, and cells with up-regulated UPR. Over the time course of cultivation, the distribution of the population into these four clusters changed. After 30 h of production, 60% of the cells producing EcPGA, which accumulated in the cells (50–70% of the product), had up-regulated UPR, but only 13% of the cells had impaired viability. A higher proportion of cells with decreased viability was observed in strains producing CaLB (20%) and TlXynA (27%). The proportion of cells with up-regulated UPR in CaLB-producing (35%) and TlXynA-producing (30%) strains was lower in comparison to the EcPGA-producing strain, and a smaller proportion of CaLB and TlXynA (<10%) accumulated in the cells. These data provide an insight into the development of heterogeneity in a recombinant P. pastoris population during a biotechnological process. A deeper understanding of the relationship between protein production/secretion and the regulation of the UPR might be utilized in bioprocess control and optimization with respect to secretion and population heterogeneity

Overview of alcohol purification methods used in beverage industry.

The article gives overview of various methods of alcohol and alcoholic beverage producers to ensure the required quality of alcohol and its possible refinement. This applies especially for the removal of fermentation by-products and accompanying substances from alcohol, which are undesirable mainly from the sensorial point of view. In addition to repeated rectification and purification, the properties of alcohol can be improved by means of adsorption methods from which the adsorption of substances on activated carbon is the most frequently used one. There exists a number of alternatives and possibilities how to increase this process. For alcohol purification, some other filtration and adsorption materials can also be used, such as techniques based on the contact of alcohol with gas, additions of various agents or ion-exchange methods.These ways of treatment and purification of alcohol can be applied especially for the production of high-quality vodka brands.The article gives overview of various methods of alcohol and alcoholic beverage producers to ensure the required quality of alcohol and its possible refinement. This applies especially for the removal of fermentation by-products and accompanying substances from alcohol, which are undesirable mainly from the sensorial point of view. In addition to repeated rectification and purification, the properties of alcohol can be improved by means of adsorption methods from which the adsorption of substances on activated carbon is the most frequently used one. There exists a number of alternatives and possibilities how to increase this process. For alcohol purification, some other filtration and adsorption materials can also be used, such as techniques based on the contact of alcohol with gas, additions of various agents or ion-exchange methods.These ways of treatment and purification of alcohol can be applied especially for the production of high-quality vodka brands

Chemical cleaning of ceramic membranes used for stillage filtration.

The main concern of this work was to find the effective membrane regeneration scheme to obtain the initial water flux of the ceramic membranes that were fouled by distillery stillage. The foulants include mainly polysaccharides, proteins, low molecular compounds and salts. The use of different cleaning strategies, temperatures, regeneration time and chemical agents with different concentrations (NaOH, H2O2, EDTA, HNO3), was realized with the aim of getting the biggest possible flux recovery. Two different cleaning strategies were experimented: inside the module by recirculation of cleaning solution or outside the module submersing the membranes in a solution of cleaning agent at initial high temperature. The effective cleaning agent was 4% sodium hydroxide solution at high temperature of 80 °C.The main concern of this work was to find the effective membrane regeneration scheme to obtain the initial water flux of the ceramic membranes that were fouled by distillery stillage. The foulants include mainly polysaccharides, proteins, low molecular compounds and salts. The use of different cleaning strategies, temperatures, regeneration time and chemical agents with different concentrations (NaOH, H2O2, EDTA, HNO3), was realized with the aim of getting the biggest possible flux recovery. Two different cleaning strategies were experimented: inside the module by recirculation of cleaning solution or outside the module submersing the membranes in a solution of cleaning agent at initial high temperature. The effective cleaning agent was 4% sodium hydroxide solution at high temperature of 80 °C

A Study of the Physiological Response of Pichia pastoris Growing in a Continuous Culture to an Induced Stress Situation

During our work, flow cytometry was used for a study of the influence of cultivation conditions on a physiological state, mainly on the viability and vitality of the recombinant Pichia pastoris strain producing ?-galactosidase. The fluorescent dyes propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol were used for the determination of the structural integrity and the membrane potential of Pichia pastoris cells, respectively. First the staining procedures were optimised and adapted for the Pichia pastoris yeast cells and later applied in a lab-scale continuous cultivation. Stained samples were analysed with the flow cytometer PAS III Partec or the epifluorescence microscope Nikon Eclipse E400. The following conditions were found to be optimum for the staining of Pichia pastoris cells: 57 ?g/ml propidium iodide in the sample, 5 min incubation time; 2 ?g/ml bis-(1,3-dibutylbarburic acid) trimethine oxonol in the sample, 20 min incubation time. The optimised staining methods were employed in a study of stress-induced physiological response to change of substrate from glycerol to methanol in a glycerol steady state growing culture of Pichia pastoris yeast. During the first 5 h of the transitional state an accumulation of methanol in the culture broth was accompanied by a decreasing concentration of biomass and an increasing amount of cells stained with propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol. After the adaptation phase the amount of cells stained with propidium iodide and bis-(1,3-dibutylbarburic acid) trimethine oxonol reached steady levels of 2% and 5%, respectively