Effect of freeze-dried immobilized cells on delignified cellulosic material in low-temperature and ambient-temperature wine making

In this article, were port on wine making by freeze-dried immobilized cells on delignified cellulosic material for ambient and low temperatures. Biocatalyst supported by freeze-dried delignified cellulosic (FDC) material recovered after the first repeated-batch fermentations the fermentation efficiency and startup, which become about equal to those of biocatalyst supported by wet delignified cellulosic material. The FDC biocatalyst was suitable for wine making at low temperatures (5–15°C), and produced wine of 12% alcoholic degree, with the main volatiles contained in the wine and reduced by a decrease in temperature. The fermentation efficiency was not affected by total acidity of must, while an increase in initial Be density improved percentages of higher alcohols and ethylacetate. The quality of the wine was validated by a preliminary taste test to be in the range of acceptable to excellent.     

Dry Red Wine Making Using Yeast Immobilized on Cork Pieces

The commercial Saccharomyces cerevisiae Uvaferme 299 wine yeast was immobilized on cork pieces to produce a biocatalyst for use in dry red wine making. The immobilized biocatalyst was suitable for clarified and non-clarified grape must fermentation at ambient and low temperatures (0–25 °C). Fermentation times were low and very low amounts of residual sugar were detected, showing suitability for dry wine production. The presence of suspended solids in the non-clarified must did not affect the activity of the immobilized cells. Complete fermentation of sugars at 0 °C was possible with immobilized Uvaferme 299, although not a cryotolerant strain, indicating a cryoprotective effect of cork. The presence of cork did not affect the composition of major volatiles with methanol and acetaldehyde kept at low levels. Reduction of amyl alcohols on total volatiles was also observed in wines fermented at low temperatures. Differences in the headspace aroma profile in wines produced by immobilized and free cells were found by GC–MS analysis, but no cork taint compounds were detected.     

Volatiles Formation from Grape Must Fermentation Using a Cryophilic and Thermotolerant Yeast

Grape must fermentation performance and volatiles formation by simultaneously cryophilic and thermotolerant yeast (strain AXAZ-1), isolated from grapes in Greece, was evaluated in a wide temperature range (5?C40°C). Yeast strain was immobilized on brewer??s spent grains (BSG) and the formed biocatalyst was introduced into a Multi-Stage Fixed Bed Tower (MFBT) bioreactor. Almost complete sugar utilization from the aforementioned biocatalyst was observed in a wide temperature spectrum, ranging from 5?°C to 37?°C, while at 40?°C residual sugar was up to 29?g/l. Time to complete fermentation with the immobilized yeast ranged from 290?h at 5?°C and 120?h at 40?°C to 25?h at 33?°C. The daily ethanol productivity reached maximum (88.6?g/l) and minimum (5.6?g/l) levels at 33?°C and 5?°C, respectively. The aroma-related compounds?? profiles of immobilized cells at different fermentation temperatures were evaluated by using solid phase microextraction (SPME) gas chromatography/mass spectrometry (GC/MS). Must fermentation resulted in a high-quality fermentation product due to the low concentrations of higher and amyl alcohols at all temperatures tested. AXAZ-1 is a very promising strain for quality wine production, as it is capable of performing fermentations of high ethanol concentration and productivities in both low and high temperatures.     

Fermentation Efficiency of Cells Immobilized on Delignified Brewers' Spent Grains after Low- and High-Temperature Thin Layer Thermal Drying

Low-cost dried yeasts immobilized on delignified brewers' spent grains for use in wine making and brewing were produced by simple thermal drying techniques. To optimize the thermal drying process, vacuum and air stream conditions were examined. Drying of thin layers of the biocatalysts was performed at low (30–38 °C) and high temperatures (40–70 °C). The fermentation efficiency of the thermally dried biocatalysts was acceptable, with immobilized cells showing a significantly higher thermotolerance compared with free cells. Immobilized cells dried at high temperatures presented slightly improved glucose fermentation efficiency compared with the low-temperature dried biocatalysts. Gas chromatography–mass spectrometry analysis of aroma volatiles of the fermented products revealed an increase of esters, lower higher alcohol formation, and significantly lower concentration of carbonylic compounds.     

Freeze-Dried Saccharomyces cerevisiae Cells Immobilized on Potato Pieces for Low-Temperature Winemaking

A biocatalyst was prepared by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on potato pieces. This biocatalyst was subjected to freeze-drying, and the effect of several protective agents and storage at 5 °C, up to 9 months, on viability and fermentative activity of yeasts cells were studied. From several protective agents tested, sodium glutamate preserved the viability of immobilized yeast cells at high levels even after 9-month storage. The freeze-drying biocatalyst was used for repeated batch fermentations of grape must at low temperatures until 5 °C. The produced wines analyzed for volatile byproducts by GC and GC/MS and the results showed that the freeze-dried biocatalysts, with sodium glutamate as protectant, produced wines with higher formation of esters than free cells and having at least similar aromatic profile to those produced by wet biocatalysts.     

Impact of High Temperature on Ethanol Fermentation by Kluyveromyces marxianus Immobilized on Banana Leaf Sheath Pieces

Ethanol fermentation was carried out with Kluyveromyces marxianus cells at various temperatures (30, 35, 40, and 45 °C). Fermentation performance of the immobilized yeast on banana leaf sheath pieces and the free yeast were evaluated and compared. Generally, ethanol production of the immobilized and free yeast was stable in a temperature range of 30–40 °C. Temperature of 45 °C restricted yeast growth and lengthened the fermentation. The immobilized yeast demonstrated faster sugar assimilation and higher ethanol level in the fermentation broth in comparison with the free yeast at all fermentation temperatures. Change in fatty acid level in cellular membrane was determined to clarify the response of the free and immobilized yeast to thermal stress. The free cells of K. marxianus responded to temperature increase by increasing saturated fatty acid (C16:0 and C18:0) level and by decreasing unsaturated fatty acid (C18:1 and C18:2) level in cellular membrane. For fermentation at 40 °C with immobilized cells of K. marxianus, however, the changes were not observed in both saturated fatty acid (C16:0) and unsaturated fatty acid (C18:1 and C18:2) level.     

Online monitoring of higher alcohols and esters throughout beer fermentation by commercial Saccharomyces cerevisiae and Saccharomyces pastorianus yeast

Higher alcohols and esters are among the predominant classes of volatile organic compounds (VOCs) that influence the quality of beer. The concentrations of these compounds are determined through a specific yeast strain selection and fermentation conditions. The effect of yeast strains on the formation of higher alcohols and esters throughout fermentations (at 20°C) was investigated. Flavour-relevant esters (ethyl acetate, isoamyl acetate, ethyl hexanoate and ethyl octanoate) and higher alcohols (isoamyl alcohol, isobutyl alcohol and phenylethyl alcohol) were monitored throughout the fermentation using proton transfer reaction–time of flight–mass spectrometry (PTR-ToF-MS) coupled with an automated sampling system for continuous measurements. Compound identification was confirmed by analysis of samples using gas chromatography–mass spectrometry (GC–MS). Results demonstrated the specific time points where variation in higher alcohol and ester generation between yeast strains occurred. In particular, the concentrations of isoamyl acetate, ethyl octanoate and isoamyl alcohol between yeast strains were significantly different over the first 2 days of fermentation; whereas, after Day 3, no significant differences were observed. The two Saccharomyces pastorianus strains produced comparable concentrations of the key higher alcohols and esters. However, the key higher alcohol and ester concentrations varied greatly between the two S. cerevisiae strains. The use of PTR–ToF–MS to rapidly measure multiple yeast strains provides new insights on fermentation for brewers to modify the sensory profile and optimise quality.     

Production of Thermostable Lipase by Thermomyces lanuginosus on Solid-State Fermentation: Selective Hydrolysis of Sardine Oil

A naturally immobilized biocatalyst with lipase activity was produced by Thermomyces lanuginosus on solid-state fermentation with perlite as inert support. Maxima lipase activities (22 and 120 U/g of dry matter, using p-nitrophenyl octanoate and trioctanoine, respectively, as substrates) were obtained after 72 h of solid culture, remaining nearly constant up to 120 h. Maxima lipase activity was found at 60 to 85 °C and pH 10. The biocatalyst was stable at 60 °C for at least 4 h of incubation and a pH from 7 to 10. Energy values of activation and deactivation of lipase were of 26 and 6.7 kJ/mol, respectively. The biocatalyst shows high selectivity for the release of the omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), during the hydrolysis of sardine oil. The EPA/DHA ratio (16:6) released by this biocatalyst was superior to that obtained with the commercial preparations of T. lanuginosus.     

Lipase-catalyzed acylation in a continuous down-flow fixed-bed reactor

The kinetic resolution of racemic 1-phenylethanol with ethyl acetate was investigated in a down-flow fixed-bed reactor operated in a continuous mode mainly at the molar ratio of 1: 3 in 400 mL toluene at 70°C. The catalytic activity of the immobilized lipase was studied by: (i) changing the flow rates, (ii) utilizing different substrate concentrations, (iii) applying step changes using ethyl acetate, ethyl benzene, acetic acid, acetophenone etc., (iv) investigating the inhibitory effect of either the desired or the stoichiometric products (R)-1-phenylethyl acetate and ethanol, respectively), (v) elucidating the effect of water on the activity and stability of the immobilized lipase. The residence time distribution and the reactor hydrodynamics were also discussed along with kinetic modelling. The results were linked to the one-pot reactions.     

Modeling of saccharide utilization in primary beer fermentation with yeasts immobilized in calcium alginate

Immobilized beer fermentation was studied using an industrial bottom-fermenting yeast strain Saccharomyces cerevisiae. The yeast cells were immobilized in 2.5% calcium alginate gel and used for brewing in a five-vessel cascade reactor. The fermentation was performed at 15°C at various flow rates. A nonstructured mathematical model was developed to simulate the performance of continuous primary fermentation of lager beer. The model was based on the following variables: maltose, maltotriose, glucose, fructose, ethanol, and cell concentration. Experimental values of these variables were determined in samples taken at regular intervals. For experimental data fitting a nonlinear regression was used. Substrate consumption was characterized by specific substrate consumption rate and saturation constant. The values of these two parameters were optimized for all four substrates. Inhibition effects of substrates and product were analyzed using various inhibition patterns. Only the inhibition effect of maltose on maltose consumption was clearly identified. A good-fitting relationship for maltose inhibition was found, and inhibition constants were calculated.     

Lipase-mediated selective acetylation of primary alcohols in ethyl acetate

An environmental friendly process to selectively acetylate primary alcohols was demonstrated. The esterification process consists of treatment of a primary alcohol in the presence of immobilized C. antarctica lipase (Novozyme-435) in ethyl acetate at room temperature. Primary alcohols were acetylated in the presence of secondary alcohols and phenols.     

Continuous wine making by γ-alumina-supported biocatalyst

The main objective of the present work was the removal of aluminum from wines produced by γ-alumina-supported yeast cells. Reagents such as Na₂CO₃, NH₄OH, albumin, and Ca(OH)₂ were used. Calcium in the presence of albumin was effective, whereas other reagents were not so effective. Because of the improved aroma and taste of distillates produced by γ-alumina-supported biocatalyst, volatile byproducts of distillates were analyzed. They were also assessed by sensory tests. Methanol, acetaldehyde, ethyl acetate, propanol-1, isobutyl alcohol, and amyl alcohols were determined in distillates. It was noted that the amounts of higher alcohols and amyl alcohols decreased as the temperature of fermentation dropped, leading to a product of improved quality and reduced toxicity.     

Production of FAME and FAEE via Alcoholysis of Sunflower Oil by Eversa Lipases Immobilized on Hydrophobic Supports

The performance of two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 immobilized in different supports was investigated. The two lipases were adsorbed on four different hydrophobic supports. Interesting results were obtained for both lipases and for the four supports. However, the most active derivative was prepared by immobilization of Eversa® Transform 2.0 on Sepabeads C-18. Ninety-nine percent of fatty acid ethyl ester was obtained, in 3 h at 40 °C, by using hexane as solvent, a molar ratio of 4:1 (ethanol/oil), and 10 wt% of immobilized biocatalyst. The final reaction mixture contained traces of monoacylglycerols but was completely free of diacylglycerols. After four reaction cycles, the immobilized biocatalyst preserved 75% of activity. Both lipases immobilized in Sepabeads C-18 were very active with ethanol and methanol as acceptors, but they were much more stable in the presence of ethanol.     

Esterification Synthesis of Ethyl Oleate in Solvent-Free System Catalyzed by Lipase Membrane from Fermentation Broth

In this study, the immobilized lipase was prepared by fabric membrane adsorption in fermentation broth. The lipase immobilization method in fermentation broth was optimized on broth activity units and pH adjustments. The viscose fermentation broth can be used with a certain percentage of dilution based on the original broth activity units. The fermentation broth can be processed directly without pH adjustment. In addition, the oleic acid ethyl ester production in solvent-free system catalyzed by the immobilized lipase was optimized. The molar ratio of ethanol to oil acid, the enzyme amount, the molecular amount, and the temperature were 1:1, 12% (w/w), 9% (w/w)(based the total amount of reaction mixture), and 30 °C, respectively. Finally, the optimal condition afforded at least 19 reuse numbers with esterification rate above 80% under stepwise addition of ethanol. Due to simple lipase immobilization preparation, acceptable esterification result during long-time batch reactions and lower cost; the whole process was suitable for industrial ethyl oleate production.     

Comparative study of amidase production by free and immobilized Escherichia coli cells

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.     

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cells in Dual Matrices

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.     

Design and Properties of an Immobilization Enzyme System for Inulin Conversion

A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40 °C for the free enzyme and 5.0–5.5 and 45–50 °C for the immobilized enzyme. The kinetic parameters of V _max and K _m were 5.24 μmol/min and 57.6 mg/mL for the free inulinase and 4.32 μmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45 °C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.     

Development of a dynamic headspace solid-phase microextraction procedure coupled to GC-qMSD for evaluation the chemical profile in alcoholic beverages

In the present study, a simple and sensitive methodology based on dynamic headspace solid-phase microextraction (HS-SPME) followed by thermal desorption gas chromatography with quadrupole mass detection (GC-qMSD), was developed and optimized for the determination of volatile (VOCs) and semi-volatile (SVOCs) compounds from different alcoholic beverages: wine, beer and whisky. Key experimental factors influencing the equilibrium of the VOCs and SVOCs between the sample and the SPME fibre, as the type of fibre coating, extraction time and temperature, sample stirring and ionic strength, were optimized. The performance of five commercially available SPME fibres was evaluated and compared, namely polydimethylsiloxane (PDMS, 100 μm); polyacrylate (PA, 85 μm); polydimethylsiloxane/divinylbenzene (PDMS/DVB, 65 μm); carboxen™/polydimethylsiloxane (CAR/PDMS, 75 μm) and the divinylbenzene/carboxen on polydimethylsiloxane (DVB/CAR/PDMS, 50/30 μm) (StableFlex).An objective comparison among different alcoholic beverages has been established in terms of qualitative and semi-quantitative differences on volatile and semi-volatile compounds. These compounds belong to several chemical families, including higher alcohols, ethyl esters, fatty acids, higher alcohol acetates, isoamyl esters, carbonyl compounds, furanic compounds, terpenoids, C13-norisoprenoids and volatile phenols. The optimized extraction conditions and GC-qMSD, lead to the successful identification of 44 compounds in white wines, 64 in beers and 104 in whiskys. Some of these compounds were found in all of the examined beverage samples.The main components of the HS-SPME found in white wines were ethyl octanoate (46.9%), ethyl decanoate (30.3%), ethyl 9-decenoate (10.7%), ethyl hexanoate (3.1%), and isoamyl octanoate (2.7%). As for beers, the major compounds were isoamyl alcohol (11.5%), ethyl octanoate (9.1%), isoamyl acetate (8.2%), 2-ethyl-1-hexanol (5.9%), and octanoic acid (5.5%). Ethyl decanoate (58.0%), ethyl octanoate (15.1%), ethyl dodecanoate (13.9%) followed by 3-methyl-1-butanol (1.8%) and isoamyl acetate (1.4%) were found to be the major VOCs in whisky samples.     

Low-temperature liquid–liquid extraction of phenols from aqueous solutions with hydrophilic mixtures of extractants

The volume ratios in acetonitrile–ethyl acetate (90 : 10, 95 : 5), acetonitrile–isopropanol–ethyl acetate (70 : 15 : 15, 80 : 5 : 15), and isopropanol–1-butanol (50 : 50) mixtures were determined. Their mixing with water (1 : 1) and storage at–10°C led to partitioning into two immiscible liquid phases without formation of the ice phase. The mixtures were shown to be useful as hydrophilic extractants in low-temperature liquidliquid extraction of phenol from aqueous solutions.     

High-temperature wine making using the thermotolerant yeast strain Kluyveromyces marxianus IMB3

Kluyveromyces marxianus IMB3 yeast cells were immobilized on delignified cellulosic material, apple, and quince separately. Both immobilized and free cells were used in high-temperature wine making, and their fermented grape must contained 3 to 4% alcohol. Semisweet wines were produced by the addition of potable alcohol to the fermented must. Preliminary sensory evaluation of the produced semisweet wines showed good flavor and aroma. The final product contained extremely low levels of higher and amyl alcohols while ethyl acetate was at levels usually present in wines. The ferment produced may be blended with other products to improve their quality.