Food Sci. Biotechnol. Vol. 18, No. 6, pp. 1358 ~ 1364 (2009) ⓒ The Korean Society of Food Science and Technology Effect of Low Intensity Pulsed Electric Field on Ethanol Fermentation and Chemical Component Variation in a Winemaking Culture He-Ryeon Min, Bo Young Jeon, Ha Na Seo, Min Ju Kim, Jun Cheol Kim1, Joon Kuk Kim, and Doo Hyun Park* Department of Biological Engineering, Seokyeong University, Seoul 136-704, Korea 1Korea Wine Academy, Seoul 135-010, Korea Abstract Electric polarity of working electrode and counter electrode was periodically switched at the intervals of 30 sec. Electric current generated by anodic and cathodic reaction of working electrode was reached to +30 and -12 mA in low intensity pulsed electric field (LIPEF). The yeast growth, ethanol production, and malate consumption in the initial cultivation time were more activated in the LIPEF than the conventional condition (CC). Polyphenol, total phenolic contents (TPC), and total flavonols (TF) were gradually decreased in all cultivation conditions during incubation for 2 weeks but antioxidation activity was not. TF was significantly lower in 3 and 4 V of LIPEF than CC and 2 V of LIPEF; however, the polyphenol, TPC, and antioxidation activity were a little influenced by the LIPEF. After ripening of the winemaking culture for 15 days, polyphenol, TPC, and TF were a little increased but the antioxidation activity was not. Keywords: winemaking culture, low intensity pulsed electric field, Saccharomyces bayanus, ethanol fermentation, antioxidation activity Introduction CO2 generated by yeast (11). CO2 is toxic to bacterial cells, especially the species depending on respiratory metabolism In an effort to improve the wine quality, some physical (12). Accordingly, the ethanol and CO2 produced by yeast techniques, such as flash evaporation and cooling time may inhibit bacterial contaminants, but do not influence controls, have been applied to the preparation of grape yeast physiology. must in the winemaking process; however, the physical All kinds of winemaking yeasts produce ethanol by a techniques can not be a critical factor affecting yeast common metabolic pathway (EMP) under anoxic conditions growth and fermentation metabolism (1). Typically, wine without exception (13) but can catalyze various sugars, quality has been largely dependent on grape species and organic acids, and even ethanol by mitochondrial metabolism yeast physiology (2,3), which are natural factors incapable under aerobic conditions (14). The critical dissolve oxygen of artificially regulating. The oxidation-reduction potential, of oxidation potential for activation of mitochondrial dissolved oxygen and bacterial contamination in the metabolism and ethanol fermentation may be difficult to winemaking cultures may be controlled by modification or induce in the winemaking culture by the existing fermentation alteration of environmental factors (4,5). Practically, the technology (15). Aeration or agitation is the general fermentation techniques to control yeast growth, fermentation techniques employed in typical bioreactors to induce rates, and chemical components have not yet been developed oxidation potential or aerobic condition. However, neither in the winemaking process. Sulfite has been applied to the aeration nor agitation can induce the critical oxidation winemaking process in order to control bacterial contamination potential to optimize the metabolism of the winemaking (6) but may inhibit some yeast strains and can induce yeast (16). unwanted chemical reaction in the winemaking culture In this study, the low intensity pulsed electric field (7,8). The grape must is naturally infected with various (LIPEF) effects on yeast growth, ethanol production, bacteria and wild yeasts during harvesting and extraction malate consumption, and variation of chemical components (9). The wild yeast that naturally contaminates the grape including polyphenol, total polyphenol, total flavonols must is difficult to control, but may be partially controlled (TF), and antioxidation activity were estimated as a part of by the inoculation of specific yeast strain, allowing the efforts to improve the wine quality and development of inoculum itself to become the predominant (10). Once new winemaking process. ethanol fermentation has begun, the oxidation-reduction potential of the winemaking culture may be maintained at the lower level than 0 V (V. Ag/AgCl) because the dissolved Materials and Methods oxygen can be purged from the winemaking culture by Chemicals All chemicals and a platinum wire used in this study were purchased from Sigma-Aldrich (St. Louis, *Corresponding author: Tel: +82-2-940-7190; Fax: +82-2-919-0345 MO, USA) except medium ingredients. E-mail: [email protected] Received April 5, 2009; Revised August 12, 2009; Electrochemical bioreactors An electrochemical bioreactor Accepted September 1, 2009 was designed to induce a LIPEF in a winemaking culture 1358 Effect of Low Intensity Pulsed Electric Field on Ethanol Fermentation 1359 company (Muju, Jeonnam, Korea), of which glucose and malate contents were analyzed with high performance liquid chromatography (HPLC) prior to use. Other chemical components were spectrophotometrically analyzed. In order to produce 12-13% of ethanol, the glucose concentration was adjusted to a final 1.5 M by the direct addition of glucose powder to the grape must. Analysis of ethanol, glucose, and malate Ethanol, glucose, and malate were analyzed via an HPLC apparatus (YoungLin, Seoul, Korea) equipped with an Aminex HPX- 87H ion exchange column (Bio-Rad, Hercules, CA, USA) and a refractive index detector (YoungLin). Samples were prepared from yeast culture via filtration with a membrane filter (pore size 0.22-µM, Satorius, Hannover, Germany) and injected directly into the HPLC injector. Concentration of target materials was determined by comparison with retention time and peak area of the chromatograms of standard materials. Analysis of polyphenols Polyphenols were determined according to Purssion blue spectrophotometric method (17). Winemaking culture was filtrated with a membrane filter (pore size 0.22-µM) and diluted with pure water in Fig. 1. Schematic structure of an electrochemical bioreactor. the range of standard curve. A 3.0 mL of 0.1 M FeCl3 in Designed to induce the pulsed electric field, in which 0.1 M HCl were added to 1 mL of the filtrate followed Saccharomyces bayanus was cultivated with grape must. immediately by timed addition of 3.0 mL of freshly prepared 0.008 M K3Fe(CN)6. The absorbance was measured on a spectrophotometer (UV-1601; Shimadzu, Tokyo, Japan) at as shown in Fig. 1. A titanium plate (thickness 0.5 mm, 720 nm after 10 min from the addition of reagents. A diameter 120 mm, VWR, Chicago, IL, USA) and platinum standard curve was prepared for expressing the results as wire (length 50 mm, diameter 0.5 mm, Sigma-Aldrich) tannic acid equivalent, i.e., amount of tannic acid (mg/L) were used as the working electrode and counter electrode, which gave a color intensity equivalent to the given by respectively. Two, 3, or 4 V of direct current (DC) polyphenols after correction for blank. electricity was charged to electrodes and electrode polarity of working and counter electrode was exchanged at the Analysis of total phenolic contents (TPC) TPC was intervals of 30 sec, by which the anodic (oxidation) and determined using the Folin-Ciocalteu colorimetric method cathodic (reduction) reaction was alternately generated on (18). The filtrated winemaking culture with a membrane working electrode. Electric current was not generated in 1 filter (pore size 0.22-µm) was properly diluted with pure V of LIPEF but the color of winemaking culture was water in the range of standard curve. In a 10 mL-conical significantly changed in 5 V of LIPEF. On the basis of tube, 6.4 mL distilled water, 0.l mL filtrate, and 0.5 mL these results, the 2, 3, and 4 V of electric intensity were Folin-Ciocalteu reagent (1:1 with water) were added and selected. The reactor volume was adjusted to 2,000 mL and mixed. After exactly 1 min, 3.0 mL of Na2CO3 (10 g/ the headspace pressure was adjusted to approximately 1.05 100 mL) was added, and the mixture allowed to stand at atm with a check valve. room temperature in the dark for 2 hr. The absorbance was read at 765 nm, and the total polyphenols concentration Cultivation and ripening condition The winemaking was calculated from a calibration curve developed using culture was incubated at 25oC and ripening temperature 10-100 mg/L of gallic acid. was adjusted to 20oC. Analysis of total flavonols (TF) TF contents were Measurement of electric pulse The electric pulse determined using the slightly modified p-dimethylamino- generated at the working electrode was measured with an cinnamalde-hyde (DMACA) method (19). The filtrated ampere-voltage meter, which was connected with a data winemaking culture with membrane filter (0.22-µm pore) acquisition system controlled by a personal computer. was properly diluted with pure water in range of standard curve. A 0.2 mL of filtrate was introduced into a 10-mL Yeast strain The yeast strain used in this study was conical tube and added 3 mL DMACA solution (0.1% in 1 Saccharomyces bayanus (EC-1118), which was purchased mol/L of HCl in methanol). The mixture was vortexed and from a professional wine yeast producer (Lallemand Inc., allowed to react at room temperature for 10 min. The Montreal, ON, Canada). The inoculum size and process absorbance at 640 nm was read against a blank prepared were consistent with the supplier’s specifications. following the sample procedure as above but without DMACA. The concentration of TF was estimated from a Grape must Grape must was presented by a winemaking calibration curve developed using 6.25-200 mg/L of catechin. 1360 H. -R. Min et al. Antioxidant activity Antioxidation activity was determined 3 V of electric intensity. Theoretically, 3.45, 14.88, and by the metal chelating capacity (20). Filtrated and diluted 21.82 mL of oxygen are generated from working electrode winemaking culture (3.8 mL) was mixed 50 µL of 10 mM at the 2, 3, and 4 V of LIPEF, respectively, for 1 hr.