Commercial Pectic Enzyme Research Articles

Pre-fermentative cold maceration in the presence of non-Saccharomyces strains: effect on fermentation behaviour and volatile composition of a red wine

Background and Aims This study evaluated the impact of pre-fermentative cold maceration (PCM), in the presence of two non-Saccharomyces yeasts, Metschnikowia pulcherrima MP 346 and Metschnikowia fructicola MF 98-3, and of a commercial pectic enzyme, on fermentation kinetics and on the volatile composition of a Sangiovese red wine. Methods and Results Sangiovese grape must was inoculated with MP 346 or MF 98-3 or treated with a pectic enzyme preparation during PCM, at 5°C for 24 or 72 h. A Control wine was produced by a pure culture of Saccharomyces cerevisiae. Both non-Saccharomyces strains affected the initial yeast population dynamics and the persistence of S. cerevisiae at the end of malolactic fermentation. Irrespective of the duration of PCM, the inoculum of Metschnikowia strains did not influence the rate of sugar consumption or the kinetics of malolactic fermentation. The volatile composition of the final wines was evaluated with solid-phase extraction, followed by GC/MS. The concentration of some terpenes and C13-norisoprenoids, nerol, geraniol, 8-hydroxy-linalool (cis) and 3-oxo-α-ionol, and of some esters, isoamyl lactate and ethyl isoamyl succinate, was higher in wines inoculated with Metschnikowia strains than in the Control and wine treated with pectic enzyme. Conclusions Metschnikowia yeast strains MP 346 and MF 98-3 affect wine volatile composition. Significance of the Study This study shows for the first time that an inoculum of Metschnikowia strains MP 346 and MF 98-3 during PCM is effective in modulating the volatile composition of a Sangiovese red wine.

Effect of winemaking techniques on polysaccharide composition of Cabernet Sauvignon, Syrah and Monastrell red wines

Background and Aims Several authors have demonstrated the interesting properties of wine polysaccharides. These compounds act as protective colloids and are able to interact with tannins and anthocyanins in wines, reducing their reactivity and increasing colour stability. Little, however, is known about the release of polysaccharides by winemaking technologies. We examine the effect of several winemaking techniques – cold prefermentative maceration, dry ice addition and grape skin freezing, and addition of two maceration enzymes – on the quantity and composition of polysaccharides extracted from the three red wine cultivars – Cabernet Sauvignon, Syrah and Monastrell. Methods and Results The molecular mass distribution and composition of polysaccharides were determined, respectively, by high-performance size-exclusion chromatography and by gas chromatography. The amount of some polysaccharide fractions extracted depended on the grape cultivar. The addition of commercial pectic enzyme preparations released a greater quantity of polysaccharides in all three cultivars studied and altered the polysaccharide composition of the Cabernet Sauvignon wine. The effect of the other treatments on the amount of polysaccharides depended on the cultivar. Conclusion This study confirms that grape cultivar and winemaking technique have a significant impact on the quantity and composition of polysaccharides extracted from grapes into wine. The concentration of polysaccharides rich in arabinose and galactose, and in rhamnogalacturonan II was greater in the Syrah wine than that in the Cabernet Sauvignon and Monastrell wines. Both enzymatic treatments and also dry ice addition had a significant influence on the polysaccharide concentration and composition of the wines made from a given cultivar, whereas cold prefermentative maceration or grape skin freezing had no effect. Significance of the Study This is the first report that shows that the polysaccharide composition of Cabernet Sauvignon, Syrah and Monastrell wines is affected by winemaking technique.

Albariño wine aroma enhancement through the use of a recombinant polygalacturonase from Kluyveromyces marxianus

The possible biotechnological application of a recombinant endopolygalacturonase of Kluyveromyces marxianus (KMPG) for the aroma enhancement of Albariño wine was studied. The addition of this enzyme to the must gives rise to a significant increase of the total compounds responsible for the aroma as opposed to the effect when using a commercial pectic enzyme. This increase also results in a significant rise of the odoriferous aglycones which are direct determinants of the aroma. Wines made by using the KMPG enzyme are characterised by a greater richness and diversity with regard to the number of aromatic compounds present, clearly differing from those obtained with a commercial pectic preparation. Based on compounds with odour activity values (OAV)>1, the wines obtained with the enzyme KMPG are richer in citric, balsamic, spicy and above all floral (violet and rose) aromas than untreated wines or wines supplemented with a commercial enzyme.

The uptake of oligogalacturonide and its effect on growth inhibition, lactate dehydrogenase activity and galactin-3 release of human cancer cells

Anti-tumour activity and membrane permeability of 1kDa oligogalacturonide (PET-pectin), prepared from citrus pectin after 24h hydrolysis, by commercial pectic enzyme produced by Aspergillus niger, on four human cell lines (HepG2, A549, Colo 205, and HEK293) and the uptake of oligogalacturonide by HEK293 cell and BALB/c mouse were investigated. PET-pectin causes a higher value of growth inhibition, lactate dehydrogenase release, and galactin-3 release in human cancer cells, as compared to the human normal HEK293 cell. There was a good correlation between growth inhibition of human cells and the uptake of rhodamine B-PET-pectin content by these cells. Additionally, almost no difference between growth inhibitions of human normal HEK293 cells cultivated with PET-pectin and pectin was found. Total pectin content in the blood of PET-pectin administrated mice increased to a maximum at 2h after oral administration, while it did not increase and change in pectin administrated mice. Our findings suggested that citrus PET-pectin can be developed as a potential dietary supplement in plant origin for cancer prevention.

Transacylation properties of pectin methyl esterase from Aspergillus niger

Transacylation reaction of citrus pectin catalyzed by pectin methyl esterase (PME) from Aspergillus niger observed by laser particle size analyzer was investigated. PME was purified by a highly methoxylation of cross-linked alcohol-insoluble solids (HM-CL-AIS) column chromatography and a subsequent Sephadex G-75 column chromatography from commercial pectic enzyme (CPE) of A. niger. The increases ofparticle size (transacylation reaction) in PME-treated pectin solution are pectin content and PME activity dependent and expressed a maximum at 0.3% pectin and 0.5 U/ml PME, respectively. PME activity (de-esterification reaction) was highly stable below 50°C but lost completely when above 70°C, while the particle size of PME-treated pectin solution decrease at temperature above 40°C. PME activity was optimum at pH 5.0, while the increase of particle size in PME-treated pectin solution reaches a maximum at pH 3.5. The different characteristics between PME from A. niger and the published plant PME reveal that there are at least two kinds of PME-catalyzed transacylation reaction, one for plant PME and the other for microbial PME. Key words: Transacylation, pectin methyl esterase, Aspergillus niger.

Antioxidant Activity and Emulsion-Stabilizing Effect of Pectic Enzyme Treated Pectin in Soy Protein Isolate-Stabilized Oil/Water Emulsion

The antioxidant activity of pectic enzyme treated pectin (PET-pectin) prepared from citrus pectin by enzymatic hydrolysis and its potential use as a stabilizer and an antioxidant for soy protein isolate (SPI)-stabilized oil in water (O/W) emulsion were investigated. Trolox equivalent antioxidant capacity (TEAC) was found to be positively associated with molecular weight (M(w)) of PET-pectin and negatively associated with degree of esterification (DE) of PET-pectin. PET-pectin (1 kDa and 11.6% DE) prepared from citrus pectin after 24 h of hydrolysis by commercial pectic enzyme produced by Aspergillus niger expressed higher α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity, TEAC, and reducing power than untreated citrus pectin (353 kDa and 60% DE). The addition of PET-pectin could increase both emulsifying activity (EA) and emulsion stability (ES) of SPI-stabilized O/W emulsion. When the SPI-stabilized lipid droplet was coated with the mixture of PET-pectin and pectin, the EA and ES of the emulsion were improved more than they were when the lipid droplet was coated with either pectin or PET-pectin alone. The amount of secondary oxidation products (thiobarbituric acid reactive substances) produced in the emulsion prepared with the mixture of SPI and PET-pectin was less than the amount produced in the emulsion prepared with either SPI or SPI/pectin. These results suggest that PET-pectin has an emulsion-stabilizing effect and lipid oxidation inhibition ability on SPI-stabilized emulsion. Therefore, PET-pectin can be used as a stabilizer as well as an antioxidant in plant origin in SPI-stabilized O/W emulsion and thus prolong the shelf life of food emulsion.

Effect of Phenolic Acid on Antioxidant Activity of Wine and Inhibition of Pectin Methyl Esterase

Antioxidant activity, malolactic fermentation and sensory evaluation of the grape must after fermentation in the presence of gallic acid and coumaric acid, as well as the inhibitory mechanism of gallic acid and coumaric acid on pectin methyl esterase (PME), were investigated. The content of malic acid and lactic acid increased 40.4% and 36.9% compared to the control when commercial pectic enzyme (CPE) was used. The increase in malic acid content was enhanced by 64.8% and 83.4%, compared to the control in the presence of CPE + Gallic acid and CPE + Coumaric acid respectively. Ferric reducing/antioxidant power (FRAP) increased in the samples with added CPE. In addition to an increase in the FRAP, antioxidant capacity was enhanced in the CPE + Gallic acid and CPE + Coumaric acid samples. No significant differences were found in the content of total anthocyanin and in the value of sensory characteristics. The content of total flavanols increased significantly in the samples with added CPE. Lineweaver-Burk plots of PME, with gallic acid or coumaric acid, indicated that gallic acid and coumaric acid were mixed inhibitors of PME.

Addition of Phenolic Acids on the Reduction of Methanol Content in Wine

Utilization of phenolic acids, including gallic acid, coumaric acid, caffic acid, cinnamic acid, and ferulic acid, for methanol reduction in wine was investigated. Enzyme activities of pectinesterase and pectin lyase decreased significantly when 0.1 mg/L of gallic acid, coumaric acid, caffic acid, cinnamic acid, or ferulic acid was added. However, no inhibition on polygalacturonase activity was observed when 0.5 mg/L of phenolic acid was added. Methanol content in commercial pectic enzyme (CPE) group increased from 11.53 +/- 1.34 to 56.67 +/- 3.75 ppm in the final products. Adding gallic acid or coumaric acid with CPE inhibited the increase of methanol production. In addition, when 0.2 mg/L of phenolic acid (gallic acid or coumaric acid) was added, the amount of total phenolic acid released from CPE + gallic acid or CPE + coumaric acid groups became higher than CPE group by approximately 466 and 539 mg/L, respectively. In conclusion, the values of lightness, red content, yellow content, total pigment, and total phenolic acid increased in the presence of gallic acid or coumaric acid with CPE, suggesting that adding gallic acid or coumaric acid into winemaking process is a potential method for reducing methanol content, improving wine quality, as well as increasing healthy compounds in wine production.

Separation and Utilization of Pectin Lyase from Commercial Pectic Enzyme via Highly Methoxylated Cross-Linked Alcohol-Insoluble Solid Chromatography for Wine Methanol Reduction

The isolation and utilization of pectin lyase (PL) from commercial pectic enzyme for methanol reduction in wine production was investigated. PL can be separated from pectinesterase (PE) and polygalacturonase (PG) on HM-CL-AIS affinity chromatography at pH 4; however, it is difficult to further distinguish PE from PG. Some desirable physicochemical properties such as transmittance, lightness, redness, and lower total pectin content are found in the external enzyme adding groups (PL, PE and PG, and pectic enzyme groups) in comparison to the control group. Methanol contents in pectic enzyme and the PE and PG groups increase from 628 +/- 13 (control group) to 3103 +/- 16 and 1736 +/- 67 mg/L ethanol in the final products, respectively. Nevertheless, the adding of PL does not cause any increase in methanol content. The results present in this study suggest that the HM-CL-AIS column is a simple, inexpensive, convenient, and effective method for PL purification. Moreover, the partial purified PL is a potential replacement of commercial pectic enzyme for pectin depolymerizing, methanol content reducing, and wine quality improving in wine production.

Comparison of some commercial pectic enzyme preparations applicable in wine technology

The preparations of pectic enzymes are used for a more efficient extraction of desirable red grape pigments and other phenol compounds which are bound in plant cells and can be faster released by the action of pectic enzymes. Moreover, they shorten the time of maceration, settling, and filtration. The results of our experiments gives a comparison of the efficiency of preparations applicable in wine technology. The best preparation was Trenolin Rot followed by Vinozym G that could shorten the time of prefermentation to about 3 days thanks to a more intensive extraction of red grape pigments. By using the enzyme preparations Gammapect AWP and Ovopres, the time of filtration was ten times shorter. Compared to the control sample, the speed of desliming was threefold and twofold faster, respectively, for Gammapect AWP and Gammapect W2L.  

High-speed pectic enzyme fractionation by immobilised metal ion affinity membranes.

Immobilised metal ion affinity polysulfone hollow-fibre membranes, with a high capacity for protein adsorption, were prepared and their utilisation for commercial pectic enzyme fractionation was studied. The pass-through fraction containing pectinlyase is useful for fruit-juice clarification without methanol production on account of pectinesterase being retained by the IDA-Cu2+ membrane.

Pectic Enzyme Treatment Effects on Quality of White Grape Musts and Wines

ABSTRACTGrapes of three white cultivars were treated with a commercial pectic enzyme preparation on an industrial scale. The effect of enzyme treatment on the following juice and wine volatiles was considered: fruity esters, terpenes, alcohols, volatile phenols (4‐vinylphenol and 4‐vinylguaiacol), hydroxycinnamate derivative esters (caftaric, coutaric, and fertaric esters), and free hydroxy cinnamic acids (caffeic, coumaric and ferulic acids). Pectic enzyme treatment increased the higher alcohols, free hydroxycinnamic acids and volatile phenols, whereas amounts of most esters, herbaceous alcohols and hydroxycinnamate derivative esters were reduced. Terpenes, 2‐phenylethanol and benzyl alcohol levels were not affected by pectic enzyme treatment. When tasted by an expert panel, differences were found in sensory characteristics, with wines from untreated judged higher quality than those from treated grapes.

Grape Pectic Enzyme Treatment Effect on White Musts & Wines Composition

ABSTRACTWhite grapes of three different cultivars (‘Macabeo’, ‘Xarel.lo’ and ‘Parellada’) were treated with the same commercial pectic enzyme preparation on an industrial scale. The effect of this treatment on must and wine composition was studied in three consecutive years (1991, 1992 and 1993). Twenty‐one parameters were determined to evaluate effects of the pecticenzyme treatment. Turbidity, galacturonic acid, total and acid polysaccharides, total and nonflavonoid phenols, proteins and meth‐anol were affected by the treatments, although this effect usually depended on the cultivar and/or year. Moreover, the effects of treatment were more marked in must than in wines. However, a considerable loss of glucosidic colloids was found on musts and wines from treated grapes. The loss of must polysaccharides depended on their initial content and were mainly due to the pectin losses. Pectin lysis was responsible for increases in galacturonic acid levels which could be used as an index in wines (within a cultivar and year) to indicate pectic enzyme treatment.

Fungal pectic enzyme fractionation by dye affinity chromatography

A dye affinity fractionation of a commercial pectic enzyme preparation is described. From nine immobilized triazinic dyes assayed, only Blue R-HE and Yellow 4R-HE were able to retain pectin lyase thus allowing the obtention of a fraction not producing methanol during fruit juice clarification. While pectinesterase did not bind to the Chromatographic matrix, the retained pectin lyase could be quantitatively eluted with a salt gradient.

Study of variables involved in fungal pectic enzyme fractionation by immobilized metal ion affinity chromatography

The effects of the pH, composition and ionic strength of the chromatographic buffer and immobilized metal on the fractionation of a commercial pectic enzyme were studied using immobilized metal ion affinity chromatography. The best performance of the system was achieved with Cu 2+ as the immobilized metal, 20 m m sodium phosphate (pH 7·0 containing 100 m m NaCl) as the adsorption buffer and 100 m m sodium acetate (pH 3·0 containing 100 m m NaCl) as the elution buffer. Under these conditions, pectinesterase was fully retained by the chromatographic matrix while the fraction passing through — containing pectin lyase — allows clarification of fruit juice without methanol production. The retained pectinesterase and the polygalacturonase contained in the commercial pectic enzyme preparation can be eluted quantitatively and applied to other industrial uses. From equilibrium adsorption isotherms, the maximum capacity of Chelating Sepharose for pectinesterase under the above conditions was 2990±190 U/ml at 20°C and 2940±170 U/ml at 30°C, while K d values were 27·1±5·3 U/ml and 57·6±9·5 U/ml, respectively. From breakthrough curves, the optimum linear flow rate was 0·80 cm/min with a commercial preparation concentration at 100 mg/ml.

Fractionation of fungal pectic enzymes by immobilised metal ion affinity chromatography

AbstractA juice‐clarifying fraction that will not produce methanol was obtained from a commercial pectic enzyme by separating the pectin lyase (EC4.2.2.10) from pectinesterase (EC3.1.1.11). This was achieved by immobilised metal ion affinity chromatography (IMAC) on Sepharose—iminodiacetic acid—Cu(II). Pectin lyase did not bind to the chromatographic matrix at pH 8.0, while pectinesterase was retained and only eluted when the pH of the buffer was brought down to 3.0. Polygalacturonase activity (EC3.2.1.15) was found in both fractions thus suggesting that IMAC can discriminate between two forms of that enzyme Both fractions have the ability to clarify apple juice, the first without producing methanol. The behaviour of each of the above enzymes in IMAC suggests that pectin lyase lacks accessible histidine residues, while pectinesterase could have one or more of them.

Medium-pressure liquid chromatography of Leozym, a pectic enzyme preparation, on ion-exchange derivatives of spheron

Leozym, a commercial pectic enzyme preparation. was subjected to medium-pressure liquid chromatography on weakly, medium, and strongly acidic cation exchangers and on weakly and strongly basic anion exchangers, and to hydrophobic chromatography on the ionogenically unsubstituted glycomethacrylate macroreticular gel of the Spheron type. The course of the gradient chromatography was examined by contiouus measurement of the absorbance of the effluent at 285 and nm, by pH and conductivity measurements on the fractions collected and by specific measurements of selected enzyme activities. Fractions containing endo- D-galacturo-nanse, pectin-lyase and pectin-esterase activity were successfully resolved. The presence of multiple forms of endo- D-galacturonanase and pectin-lyase in the preparation examination was shown. The technological importance of these findings is briefly discussed.

A Comparison of Activity and Effects of Two Commercial Pectic Enzyme Preparations on White Grape Musts and Wines

White grape musts of eight different varieties were treated with pectic enzyme preparations. These treatments resulted in increases in total juice yields, clarity of the wine, filterability, methanol production, wine quality, browning capacity and amounts of solids that settled. Standardizing the preparations on the basis of polygalacturonase activity showed that differences in parameters such as browning capacity, methanol production, and juice yield are due to the activity of the preparation on the basis of its apple juice depectinizing activity.

The extraction of the enzyme degrading the limonin precursor in citrus albedo

AbstractOptimum conditions for extracting the enzyme degrading the limonin precursor in citrus albedo involved treatment of the macerated albedo with a commercial pectic enzyme. The release of the enzyme from the cells of the albedo was accompanied by a considerable loss of stability, and the instability of the enzyme once dissociated from the albedo cells, together with its low activity in a highly pectinous source, accounts for the failure of previous attempts at its extraction. A method for proximate assay of enzyme actiyity was developed to monitor the extraction process and to compare the concentrations of enzyme activity in various albedo samples.

Methanol in Wines and Musts: Source and Amounts

An analysis of the methanol content of wines was conducted using gas chromatography. Methanol production was followed after complete maceration of the grapes, during fermentation, and in several wines treated with commercial pectolytic enzyme preparations. A survey of some varietal wines was also made. Following maceration there was no detectable change in the methanol content of the grapes within about the first 30 minutes or any large change up to 36 hours for grapes stored at 32 or 70 F (room temperature). However, for grapes stored and macerated at 100 F methanol content increased significantly after 48 hours. There was no significant difference in maceration effects between a red and a white grape. The methanol produced during fermentation soon reached a plateau in white varieties of which only the free-run juice was fermented. Red varieties, however, with skin contact produced methanol continuously throughout fermentation. A temperature difference between 60 and 70 F made little difference in the methanol content of the wines. The use of commercial pectic enzyme preparations does increase the methanol content of wines, though more so for red varieties. Though the use of these preparations increased the methanol content proportionately more for white varieties, the final methanol content is less than that for similarly treated red varieties. The use of these preparations increased the methanol content of red wines during the early stages of fermentation.