Modelling of low temperature wine-making,using immobilized cells

A new biocatalyst was prepared by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on whole corn grains. This biocatalyst was used for 30 repeated batch fermentations of glucose and grape musts at various temperatures. The biocatalyst retained its operational stability for a long period and it proved able to produce dry wines of fine clarity, even at extremely low temperatures (5 °C). The produced wines were analyzed for volatile byproducts by GC and GC–MS. The results showed an increase in the number and amount of esters produced when immobilized cells were used. In addition, an increase in the percentages of esters and a decrease in those of alcohols with the decrease of fermentation temperature were observed. Finally a model, appropriate for describing sugar consumption and the simultaneous ethanol formation by immobilized cells, was proposed. The results of this model fit almost perfectly with the experimental data.     

Winemaking by barley supported yeast cells

A new biocatalyst was prepared by immobilisation of Saccharomyces cerevisiae AXAZ-1 yeast cells on whole barley grains. This biocatalyst was used for 30 repeated batch fermentations of glucose and grape must at various temperatures. The biocatalyst retained its operational stability for a long period and it proved capable to produce dry wines of fine clarity even at extremely low temperatures (5 °C). The produced wines were analysed for volatile byproducts by GC and GC–MS. The results showed an increase in the number and amount of esters produced when immobilised cells were used. In addition, an increase in the percentages of esters and a decrease in those of alcohols with the decrease of fermentation temperature were reported.     

Freeze-dried wheat supported biocatalyst for low temperature wine making

A new biocatalyst was prepared by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on whole wheat grains. This biocatalyst was subjected to freeze-drying and the effects of several protective agents and storage at 5 °C on viability and fermentative activity of yeasts cells were studied. Glycerol was the best protective agent that preserved the viability of immobilized yeast cells even after 9 month storage. After freeze-drying the biocatalyst was used for repeated batch wine making at extremely low temperatures until 5 °C. The produced wines were analyzed for volatile byproducts by GC and GC/MS and the results showed that the freeze-dried immobilized biocatalysts, with glycerol as protectant, produced wines with higher formation of esters than free cells, having at least similar aromatic profile to those produced by wet biocatalysts.     

Apple pieces as immobilization support of various microorganisms

The biocatalyst prepared by immobilization of Saccharomyces cerevisiae strain AXAZ-1 on apple pieces was used for fermentation of grape must both in batch and continuous system. The immobilized yeast showed an important operational stability without any decrease of its activity. In continuous process, the bioreactor was operated for 95 days without any diminution of the ethanol productivity. Concentrations of higher and amyl alcohols were low indicating an improved quality product.Apple pieces were also used as support for immobilizing Kluyveromyces marxianus IMB3 for high-temperature (45 °C) wine-making. The fermented grape must contained 3-4% alcohol and semi-sweet wines were produced by the addition of potable alcohol.Preliminary sensory evaluation of the produced wines ascertained the fruity aroma and the overall improved quality compared to other commercially available products.Lactobaccillus casei cells immobilized on apple pieces were used for probiotic, fermented milk and lactic acid production. Apple-supported L. casei used for successive fermentation batches of whey proved to be very effective and suitable for food-grade lactic acid production. The immobilized biocatalyst was also used for milk fermentation and was able to ferment after storage for 15, 98 and 129 days at 4 °C, while no infection was reported during storage periods.     

Hydrolysis of sucrose by invertase immobilized on nylon-6 microbeads

A commercial extracellular invertase (EC 3.2.1.26) from Saccharomyces cerevisiae has been inmobilized by covalent bonding on novel microbeads of nylon-6 using glutaraldehyde. The enzyme was strongly bound on the support, immobilized with an efficiency factor of 0.93. The biocatalyst showed a maximum enzyme activity when immobilized at pH 5.0, but optimum pH activity for both immobilized and free invertases was 5.5. The optimum temperatures for immobilized and free enzymes were 60 and 65 °C, respectively. Kinetic parameters were determined for immobilized and free invertases: V_max values were 1.37 and 1.06 mmol min⁻¹ mg⁻¹, respectively. The K_m and K_i values were 0.029 and 0.71 M for immobilized invertase and 0.024 and 0.69 M for free invertase. It was found that the thermal stability of the immobilized invertase with regard to the free one increased by 25% at 50 °C, 38% at 60 °C and 750% at 70 °C. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous sucrose hydrolysis. The effects of two different sugar concentrations and three flow rates on the productivity of the reactor and on the specific productivity of the biocatalyst were studied. The system demonstrated a very good productivity up to 2.0 M sugar concentration, with conversion factors of 0.95 and 0.97, depending on sucrose concentration in the feeding. This approach may serve as a simple technique and can be a feasible alternative to continuous sucrose hydrolysis in a fixed bed reactor for the preparation of fructose-rich syrup.     

Comparative study of batch and continuous multi-stage fixed-bed tower (MFBT) bioreactor during wine-making using freeze-dried immobilized cells

A freeze-dried immobilized biocatalyst produced by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on gluten pellets and subsequent freeze-drying was used in a multistage fixed-bed tower (MFBT) bioreactor for batch and continuous wine-making. The MFBT bioreactor resulted in higher alcohol productivity compared to fermentations carried out in a packed bed (PB) bioreactor and showed an important operational stability and no decrease in activity, even at low fermentation temperature (5 °C) and after storage for 6 months at 4 °C. The production of amyl alcohols proved to be temperature dependent and was significantly reduced at low temperatures. Re-activation of the freeze-dried immobilized cells after storage for 6 months resulted in further decreased content of amyl alcohols. The SPME GC/MS analysis of volatile compounds revealed no significant differences in the wines produced by MFBT and PB bioreactors, while the preliminary sensory evaluation ascertained the overall improved quality of the produced wines. Potential industrial application of MFBT bioreactor is also assessed and discussed.     

Amine concentrations in wine stored in bottles at different temperatures

Excessive concentrations of amines are undesirable in wine because they could be toxic or have a negative effect on aroma. Wine storage temperature has a decisive influence on product quality because a rise in temperature augments reactions within the wine. The aim of this study was to examine the evolution of biogenic and volatile amines during storage of wines in bottle at different temperatures. To do so, Chardonnay wine was stored in bottles at 4, 20 and 35 °C, during 105 days. The results showed that wine storage temperature had only a slight effect on the concentration of amines. The concentration of histamine stands out, as it was higher in the wine kept for 105 days at 20 °C than at the two more extreme temperatures of 4 and 35 °C. The formation or degradation of amines was mainly produced during the first 45 days of wine storage for all the temperatures under study.     

Low‐temperature wine‐making using yeast immobilized on pear pieces

A biocatalyst was prepared by the immobilization of Saccharomyces cerevisiae AXAZ‐1 yeast cells on pear pieces and tested for grape must fermentation in both batch and continuous conditions. The immobilized yeast cells were stable and active even at low temperatures (<10 °C). Wine production under batch fermentation at 8 °C was completed within 15 days while at 3 °C it took 36 days. In continuous fermentation, the bioreactor was operated for 33 days, then stored for 12 days at 10 °C, and re‐run for another 15 days without any diminution of the ethanol productivity. Total acidity of the produced wines remained within the ranges usually observed in dry wines, while volatile acidity was found in rather increased levels. The concentrations of higher alcohols (1‐propanol, isobutyl alcohol and amyl alcohols) were relatively low, while ethyl acetate was detected at up to 118 mg l^?1, contributing to the fruity aroma of the wines produced. Preliminary sensory evaluations carried out in the laboratory indicated the fine quality of the produced wines. Copyright © 2004 Society of Chemical Industry     

Design and characterisation of an enzyme system for inulin hydrolysis

The optimal conditions for inulin hydrolysis using a commercial inulinase preparation, either free or immobilised in activated Amberlite were established by factorial design and surface response methodology. The immobilised biocatalyst displayed highest activity at pH 5.5 and 50 °C, whereas the optimum pH for the free form was slightly more acidic (4.5), and the optimum temperature was a little higher (55 °C). The model system estimated optimal pH and temperature values of 5.4 and 52 °C for the immobilised system and 4.9 and 56 °C for the free system. Michaelis–Menten type kinetics adequately described both free and immobilised bioconversion systems, which were evaluated under the respective optimal pH and temperature conditions. The use of a non-linear regression method for the determination of the kinetic parameters provided a best fit to the experimental data, as compared to a conventional Lineweaver–Burk linearisation. The K_m for inulin of the free biocatalyst was 153 g l⁻¹ at 55 °C and pH 4.5, whereas the apparent K_m for inulin of the immobilised biocatalyst was 108 g l⁻¹ at pH 5.5 and 50 °C. The reutilisation of the immobilised biocatalyst throughout consecutive batches was evaluated. A significant decrease of enzyme activity was observed in the first two batches, after which the system exhibited significant stability. The low cost of the support, the stability of the immobilised biocatalyst towards pH and temperature and its high affinity for the substrate suggests its potential for inulin hydrolysis.     

The effect of heat and light on the composition of some volatile compounds in wine

HS-SPME–GC–MS analysis of Aglianico del Vulture red wine from Southern Italy showed the presence of ethyl acetate, 3-methyl-1-butanol, 3-methylbutyl acetate, ethyl hexanoate, diethyl butanedioate, ethyl octanoate, and ethyl decanoate. Thermal treatment for 24 h in the 20–60 °C range showed that for most of the compounds analysed the simple transition from 20 to 30 °C induces a significant modification on the composition of volatile compounds. A sharp decrease was observed for ethyl acetate, diethyl butanedioate, and 3-methylbutyl acetate; a moderate decrease was observed for ethyl hexanoate, while an increase was observed for 3-methyl-1-butanol, ethyl octanoate, and ethyl decanoate. The temperature of 40 °C induces the maximum evolution of this type of compound. Higher temperatures induce a reduction of volatile compounds in wine. Irradiation of Barolo (red wine, Northern Italy), Amarone (red wine, Northern Italy), and Brunello di Montalcino (red wine, Central Italy), at 20 °C was performed with a 13 W neon lamp. 3-Methyl-1-butanol was affected by the irradiation. In the case of Barolo and Amarone, we observed a moderate increase, while, for Brunello di Montalcino, a decrease was observed. In the same way, esters were degraded during irradiation. In the red wine one or more components (phenols?) are able to protect esters from photodegradation. In diethyl butanedioate and ethyl hexanoate, octanoate, and decanoate we observed an initial increase of the concentration of the esters in some cases.     

Low temperature brewing using cells immobilized on brewer’s spent grains

A biocatalyst, prepared by immobilization of the psychrotolerant yeast strain Saccharomyces cerevisiae AXAZ-1 on delignified brewer’s spent grains, was used for brewing at very low temperatures, resulting in beers with fine clarity, excellent quality and mature character after the end of primary fermentation. Fermentation times were low (only 20 days at 0 °C), while ethanol and beer productivities were high, showing suitability of the biocatalyst for very low temperature brewing. Diacetyl (61–167 ppb), 2,3-pentadione (32–109 ppb) and DMS (11–37 ppb) concentrations in all the green beers were low (the lowest values being those found in beers produced at 0–5 °C). GC/GC–MS analysis showed significant quantitative differences in the composition of aroma volatiles, revealing an impact of fermentation temperature on organoleptic qualities. The increased productivities and fine quality of the products, with low vicinal diketone, DMS and amyl alcohol concentrations, may allow elimination of maturation time, resulting in reduction of production and investment costs.     

Fermentative behavior of Saccharomyces strains during microvinification of raspberry juice (Rubus idaeus L.)

Sixteen different strains of Saccharomyces cerevisiae and Saccharomyces bayanus were evaluated in the production of raspberry fruit wine. Raspberry juice sugar concentrations were adjusted to 16°Brix with a sucrose solution, and batch fermentations were performed at 22 °C. Various kinetic parameters, such as the conversion factors of the substrates into ethanol (Y_p/s), biomass (Y_x/s), glycerol (Y_g/s) and acetic acid (Y_ac/s), the volumetric productivity of ethanol (Q_p), the biomass productivity (P_x), and the fermentation efficiency (E_f) were calculated. Volatile compounds (alcohols, ethyl esters, acetates of higher alcohols and volatile fatty acids) were determined by gas chromatography (GC-FID). The highest values for the E_f, Y_p/s, Y_g/s, and Y_x/s parameters were obtained when strains commonly used in the fuel ethanol industry (S. cerevisiae PE-2, BG, SA, CAT-1, and VR-1) were used to ferment raspberry juice. S. cerevisiae strain UFLA FW 15, isolated from fruit, displayed similar results. Twenty-one volatile compounds were identified in raspberry wines. The highest concentrations of total volatile compounds were found in wines produced with S. cerevisiae strains UFLA FW 15 (87,435 μg/L), CAT-1 (80,317.01 μg/L), VR-1 (67,573.99 μg/L) and S. bayanus CBS 1505 (71,660.32 μg/L). The highest concentrations of ethyl esters were 454.33 μg/L, 440.33 μg/L and 438 μg/L for S. cerevisiae strains UFLA FW 15, VR-1 and BG, respectively. Similar to concentrations of ethyl esters, the highest concentrations of acetates (1927.67 μg/L) and higher alcohols (83,996.33 μg/L) were produced in raspberry wine from S. cerevisiae UFLA FW 15. The maximum concentration of volatile fatty acids was found in raspberry wine produced by S. cerevisiae strain VR-1. We conclude that S. cerevisiae strain UFLA FW 15 fermented raspberry juice and produced a fruit wine with low concentrations of acids and high concentrations of acetates, higher alcohols and ethyl esters.     

Influence of temperature on continuous high gravity brewing with yeasts immobilized on spent grains

Flavor compounds’ formation and fermentative parameters of continuous high gravity brewing with yeasts immobilized on spent grains were evaluated at three different temperatures (7, 10 and 15 °C). The assays were performed in a bubble column reactor at constant dilution rate (0.05 h⁻¹) and total gas flow rate (240 ml/min of CO₂ and 10 ml/min of air), with high-gravity all-malt wort (15°Plato). The results revealed that as the fermentation temperature was increased from 7 to 15 °C, the apparent and real degrees of fermentation, rate of extract consumption, ethanol volumetric productivity and consumption of free amino nitrogen (FAN) increased. In addition, beer produced at 15 °C presented a higher alcohols to esters ratio (2.2–2.4:1) similar to the optimum values described in the literature. It was thus concluded that primary high-gravity (15°Plato) all-malt wort fermentation by continuous process with yeasts immobilized on spent grains, can be carried out with a good performance at 15 °C.     

Electronic nose to study postharvest dehydration of wine grapes

Montepulciano grapes were dehydrated at 10 and 20 °C, 45% RH and 1–1.5 m/s of air flow. Samplings were performed at 10%, 20%, 30%, and 40% of weight loss (wl). TSS (total solids content) increased up to 43° and 36° Brix at 20 and 10 °C, respectively, in 27 and 48 days. A straight regression line between M (kg water/kg dry matter) loss and days of dehydration with R² values, equal to 0.97, was found but an angular coefficient of −0.048 and −0.038, respectively at 20 and 10 °C indicated a different rate of weight loss. Volatiles compounds profile changed significantly with the temperature during the dehydration process. Anaerobic metabolites such as ethanol, acetaldehyde, and ethylacetate were much higher at 20 °C than at 10 °C and rose progressively, while at 10 °C, the significant rise was observed between 30% and 40% weight loss. Alcohols and esters prevailed at 20 °C while at 10 °C larger abundances of aldehydes and terpene alcohols are observed. PCA analysis of GC/MS confirmed the volatiles compounds separation between the samples held at 20 and 10 °C. A progressive evolution of the profile is observed for the samples held at 20 °C while those held at 10 °C show a change of volatiles compounds profile only at 40% wl. Loadings analysis show that aldehydes and terpenols are oriented towards the direction of 10 °C samples while alcohols and esters in that of 20 °C samples. The behaviours of GC/MS data were partially corroborated by the data of electronic nose. Electronic nose was able to follow the progressive change of aroma profile at 20 °C while at 10 °C grouped the responses obtained at 10%, 20%, 30% wl all together but at 40% wl, it discriminated the aroma response. This result opens a window on the potential use to monitor commercially the grape dehydration.     

Low temperature,continuous wine ‐ making by immobilized cells: A comparative study of the effect of temperature on volatile by ‐ products

Abstract

A comparison study concerning the formation of methanol, ethylacetate, propanol‐1, isobutanol and amyl alcohols (mixture of 2‐ methylbutanol‐1 and 3‐methylbutanol‐1) in continuous wine‐making by γ‐alumina, kissiris and alginates supported biocatalysts is performed in this investigation. The effect of temperature was also examined. Methanol content was not affected by the support, process and temperature, but the concentrations of the other above compounds were reduced by the reduction of the temperature in all studied supports. The percentage content of ethyl acetate in the total volatile determined plus methanol was higher in the case of immobilized cells than in free cells. Kissiris supported biocatalyst increased this parameter but the other and free cells decreased it. Propanol‐1, isobutyl‐alcohol and amyl alcohols as percentage on total volatiles determined plus methanol were less than those of free cells for all supports and temperatures studied. The effect of the improvement of the formation of volatiles on quality of the wine was examined by a taste test.     

Immobilization and stabilization of pectinase by multipoint attachment onto an activated agar-gel support

Pectinase was immobilized on an activated agar-gel support by multipoint attachment. The maximal activity of immobilized pectinase was obtained at 5 °C, pH 3.6, with a 24 h reaction time at an enzyme dose of 0.52 mg protein/g gel, and the gel was activated with 1.0 M glycidol. These conditions increased the thermal stability of the immobilized pectinase 19-fold compared with the free enzyme at 65 °C. The optimal temperature for pectinase activity changed from 40 to 50 °C after immobilization; however, the optimal pH remained unchanged. The immobilized enzyme also exhibited great operational stability, and an 81% residual activity was observed in the immobilized enzyme after 10 batch reactions.     

Effect of cultural system and storage temperature on antioxidant capacity and phenolic compounds in strawberries

The effects of cultural systems and storage temperatures on antioxidant enzyme activities and non-enzyme antioxidant components in two cultivars (‘Earliglow’ and ‘Allstar’) of strawberries were investigated. Fruit samples were hand-harvested from organic and conventional farms in Maryland, USA, and were stored at 10, 5 and 0 °C. The results from this study showed that strawberries grown from organic culture exhibited generally higher activities in antioxidant enzymes. Moreover, the organic culture also produced fruits with higher level of antioxidant contents. Strawberries stored at higher temperature (10 °C) had higher activities of antioxidant enzymes and antioxidant capacities than those stored at lower temperatures (0 or 5 °C), in both organic and conventional cultural systems. In conclusion, strawberries produced from organic culture contained significantly higher antioxidant capacities and flavonoid contents than those produced from conventional culture, and even though low storage temperatures retarded decay, they also reduced the increase in antioxidant activities.     

Combined effects of freezing rate, storage temperature and time on bread dough and baking properties

This study compares the effects of freezing temperature and rate as well as storage temperature and time on the quality of frozen dough. Yeasted bread dough was frozen using four freezing rates (19-69 °C/h), then stored at −10, −20, −30, or −35 °C for up to 180 days. Dough strength diminished with longer storage time and higher storage temperatures. Cryo-SEM showed that dough stored at −30 and −35 °C had the least damaged gluten network. NMR studies showed that more rapidly frozen dough, and that stored at lower temperatures had lower transverse relaxation (T₂) times (9-10 ms). However, dough stored at −20 °C displayed the highest yeast activity among samples. Bread loaf volume decreased with storage time, and bread made from dough stored at −20 °C showed the highest loaf volume. Breads produced from −30 and −35 °C stored dough displayed less change in the texture profile during storage as well as less change in T₂ values. Response surface analysis showed that optimal properties occurred at freezing rates of around 19-41 °C/h and storage temperatures of −15 to −20 °C.     

黄酒固定化发酵过程及香气物质形成的研究

将黄酒酵母用海藻酸钙包埋制成固定化酵母,研究游离酵母与固定化酵母的黄酒发酵过程理化指标和香气物质生成量的差异,结果表明:经过主发酵5 d(28℃),后发酵15 d(15℃),固定化酵母发酵过程的酒精度、总糖、总酸变化曲线与游离发酵过程曲线差异不大,固定化酵母发酵性能稳定,杂菌污染可控。用气相色谱(GC)分析乙酸乙酯等9种黄酒特征性香气物质,发现游离发酵黄酒正丙醇、异丁醇、异戊醇、β-苯乙醇的总高级醇含量为680.91 mg/L,固定化发酵第1,2,3,4,5批黄酒的总高级醇含量分别为633.63,649.79,658.17,637.63,614.68 mg/L,说明固定化发酵有利于控制黄酒中的高级醇含量。固定化发酵酯类总量高于游离酵母,且随着固定化酵母发酵批次的增加而递增,即:固定化第5批(115.90 mg/L)>第4批(90.81 mg/L)>第1~3批(80.10~81.81 mg/L)>游离酵母(55.66 mg/L),表明固定化发酵使黄酒的酯类香气愈加浓郁。根据香气阈值计算的固定化发酵酿得的黄酒的香气不比游离酵母酿得的黄酒差,固定化酵母发酵生产黄酒有商业化应用的前景。     

Immobilization of pepsin on chitosan beads

In this study, chitosan beads were prepared by using a cross-linking agent and the resulting beads were employed in immobilization process. Studies on free and immobilized pepsin systems for determination of optimum temperature, optimum pH, thermal stability, pH stability, operational stability, storage stability and kinetic parameters were carried out. The optimum temperature interval for free pepsin and immobilized pepsin were 30–40 and 40–50 °C, respectively. Free and immobilized pepsin showed higher activity at pH 2.0–4.0. Apparent K_m = 12.0 g L⁻¹ haemoglobin (1.56 mM tyrosine) and V_max = 5220 μmol (mg protein min)⁻¹ values were obtained for free pepsin, while apparent K_m = 20.0 g L⁻¹ haemoglobin (2.16 mM tyrosine) and V_max = 2780 μmol (mg protein min)⁻¹ values were obtained for immobilized pepsin. Thermal stability and storage stability of immobilized pepsin were higher than that of free pepsin. Milk clotting activity was used for evaluation of the applicability of pepsin immobilization to industrial process. Optimum milk clotting temperature was found as 40 °C for free pepsin and 50 °C for immobilized pepsin.