Tomoko Murakami Midori Kasai Michiyo Kumagai Fumiko Konishi Keiko Hatae In preparation of fruit liqueur, the effects of freezing pretreatment on the dissolution of ascorbic acid and polyphenol from the fruits to the liquor were examined. The freezing pretreatment of lemon and kiwifruit soaked in a lution of components such as ascorbic acid and polyphenol. About 70% of the ascorbic acid in fruits remained in the liquor after soaking for 4 days. Addition of sugar to the fruit in the liquor pretreated with freezing suppressed the dissolution of ascorbic acid and polyphenol. Both soaking in alcohol and freezing pretreatment resulted in lower impedance values and disappearance of a Cole-Cole plot, suggesting serious damage to the cell structure of 1 cubic cm of apple sample. Especially, the Cole-Cole plot completely disappeared after 3 h soaking when the fruit was soaked in 35% alcohol. It was suggested that alcohol affected mainly the dissolution of the fruit components. Key word fruit liqueur, freezing pretreatment, alcohol, ascorbic acid, polyphenol, total acidity INTRODUCTION Fruit liqueur is a kind of mixed fruit liquor prepared by soaking the fruits in spirits liquor containing 35% alcohol. Generally, it takes from one to three months to make a fruit liqueur though the time differs from one fruit to another. Recently, it was thought that the feasibility of freezing pretreatment might be useful in the production of fruit liqueur because the process could be performed at home since freezers were widely available everywhere at this time. In the usual way of producing this product, no freezing treatment no study on the dissolution of components freezing pretreatment has been reported. is applied, and so far, in fruits with In the previous study on the effect of freezing pretreatment on Azuki beans, it was found that the cooking time was shortened and a rise in the L value and a decrease in the "a" and "b" values in An's color was observed (Murakami et al., 2002 a, b). This shows that the components of foods are easily influenced by the freezing pretreatment, as manifested in the change in color during boiling. The main fruit components dissolved in the liquor in the making of fruit liqueur include polyphenol and ascorbic acid. It has been reported that fruit wines contain anthocyanins and phenols (Mazza et al., 1999), free amino acids (Tominaga et al., 2001), volatile compounds, organic * Kushiro Campus, Hokkaido University of Education ** School of Human Life and Environmental Science, Ochanomizu University *** Faculty of Culture and Education, Saga University **** Faculty of HomeEconomics, Wayo Women's University inquiry Kushiro Campus, Hokkaido University of Education 1-15-55 Shiroyama, Kushiro-shi, Hokkaido, 085-8580 TEL/FAX 0154(44)3365 acids and sugars as well as ascorbic acid (AsA). The level of ascorbic acid in the kiwifruits is much smaller than that of other organic acids such as citric acid (Soufleros et al., 2001). The amount of ascorbic acid contained in the fruit liqueur had never been reported, because it had been thought that a long soaking time could greatly decrease the total amount of ascorbic acid in the fruit liqueur. However, ascorbic acid in the fruit liqueur could possibly be retained if the soaking time was shortened by employing other means such as prefreezing in the preparation of fruit liqueur. This paper investigated the effect of freezing pretreatment in the preparation of fruit liqueur. The influence of freezing pretreatment on the dissolution of ascorbic acid and polyphenol from fruits to the liquor was examined, and the role of sugar on the dissolution of fruit components during the soaking of the fruits in alcohol was studied. Impedance analysis was carried out (Ohnishi et al., 2002) to examine the relation of fruit liqueur to component dissolution. MATERIALS AND METHODS 1. Sample preparation Lemons and kiwifruits were bought in a retail store (Tokyo, Japan) on the day of the experiment. Fruit liqueur was prepared following the method described in a cooking book (Fujimaki, 2001). Lemons were peeled and cut into round slices with the thickness of 10 mm. Kiwifruits were peeled and cut in half crosswise. Each fruit sample of 300 g was put into a glass jar disinfected with boiling water and infused with 2-fold "White liquor" (Japanese distilled spirits containing 35% alcohol, w / v), and 44 (266)
150 g of sugar was added, which was half the weight of the fruits. This was to determine the influence of sugar. 2. Freezing and soaking treatment The mixture of fruit and liquor was frozen in a freezer (Yamato Science Co., CF-21 SD, Tokyo, Japan) at - 20 C for 24 h. After freezing pretreatment, the samples were left in a room with a constant temperature of 20 C and 60 % humidity for 4 days. Though the fruits were frozen when removed from the liquid, the alcohol remained unfrozen, and this was defined as "0 soaking day" in this study. In the case of no freezing pretreatment, the mixture of fruit and liquor was soaked in a room with a constant temperature of 20 C and 60% humidity for 4 days ; in addition, soaking was extended for 90 days following the method written in a cooking book (Fujimaki, 2001). After soaking, the fruits were separated from the liquid with a separator (the liquid was expressed as fruit liqueur). The fruit liqueur obtained was filtered with filter paper or a membrane filter of 0.45,um and was then analyzed. 3. Measurement of freezing and thawing temperature Glass jars with the mixture of fruits and liquor were placed in the freezer at - 20 C. To measure the temperature of the samples, a thermo-couple attached to a data collector (Anritsu Meter Co., AM-7002, Tokyo, Japan) was inserted into the fruits and in the liquor, and the freezing temperature was measured at intervals of 5 minutes for 24 h. The frozen sample was thawed in a room with a constant temperature of 20 C, 60% humidity, where the temperature was measured at intervals of 5 minutes until the sample reached the thawing stage at room temperature. 4. Measurement of the VC content The Vitamin C (VC) content was examined by an HPLC system (Shimadzu Co., CLASS-VP, Kyoto, Japan) with a post column conductor (Yasui and Hayashi, 1993). The total of AsA (reduced AsA) and DHA (dehydrogenated AsA) was measured to calculate the total amount of VC. The fruit liqueur of lemon and kiwifruit was diluted 5-fold with 5% metaphosphoric acid. Separation was achieved on a Shim-pack SCR-101 N (7.9 mm cb x 30 cm) column with a guard column attachment. The column temperature was maintained at 40 C. A flow rate of 1.0 ml / min was used. The solvent used was aqueous sodium oxalate buffer solution (ph 3.8, 10 mm) containing 1 mm EDTA 2 Na. Analyses were performed using an SPD-M 10 Avp photodiode array UV detector. The reaction reagent used was sodium hydroxide (100 mm) containing 50 mm sodium boronhydride at a flow rate of 0.5 m// min. In the case of fresh fruits, 10 g of fresh fruit was homogenized with metaphosphoric acid and the homogenate was centrifuged to separate the fruit and the fruit liqueur. 5. Measurement of polyphenol content The polyphenol content was measured according to the method of Ishida (1993), and the amount of tannin which was used for the standard curve was calculated. The fruit liqueur was diluted 10-fold with deionized water. To 3 ml of the samples, Folin-Ciocalteau reagent was added, allowed to stand for 3 min, 3 ml of 10% Na2CO3 added, and the mixture was then left to stand for 60 min. The absorbance of the samples was recorded at 750 nm using a spectrophotometer (Shimadzu Co., UV-2100 PC, Kyoto, Japan). In the case of the fresh fruits, a 5 g sample was homogenized with 25 ml of 80% ethyl alcohol and filtered to separate the fruits from the fruit juice, and the juice was adjusted to a final volume of 50 ml. 6. Measurement of titratable acidity The fruit liqueur was diluted 5-fold with deionized distilled water. Titratable acidity was measured by titrating 10 ml samples with 0.1 N sodium hydroxide. Acidity was converted to citric acid % equivalent. 7. Measurement of the color difference The color of the fruit liqueur was measured by a spectrophotometer The data were expressed the color difference between (Minolta Co., CM-3500 d, Tokyo, Japan). as L, "a" and "b' values, and each sample and the white liquor was calculated using the values of L, "a" and "b". 8. Impedance analysis Impedance was measured by an LCR meter (Hewlett- Packard, HP 4285 A, Japan). Two electrodes of platinum wire 0.3 mm in diameter were inserted in the center part of the sample with an interval of 7 mm between the wires. It was difficult to insert the platinum electrodes in the center part of the lemon and kiwifruit used in this study; therefore, apple flesh was used in a model experiment because it is easily molded to a uniform size. Twenty five grams of peeled apples that were cut into cubes (10 x 10 x 10 mm) with a kitchen knife was added to 50 ml 35% ethanol solution and left for 24 h in a freezer at - 20 C ; after that, the samples were left for 24 h in a room with a constant temperature of 20 C and 60% humidity. In another sample preparation, 25 g of peeled apples was immersed in 50 ml of 35% ethanol solution, and one mixture was frozen and the other was not. Moreover, another 25 ml of peeled apple was immersed in 50 ml of (267) 45
J Cookery Sci. Jpn. Vol. 40 No. 4 (2007) 35% ethanol and sugar added at 25% of the amount of ethanol. One mixture was frozen and the other was not. Impedance was measured every 30 minutes for 4 h. The impedance of these samples was measured and compared with that of fresh apple. The frozen sample was measured after thawing. Resistance value (R) and reactance value ( X) in a frequency range from 20 Hz to 50,000 Hz were measured using an LCR meter. The impedance (Z) was calculated from the following equation: RESULTS AND DISCUSSION 1. Freezing and thawing temperature Fig. 1 shows the temperature in the center part of the sarcocarp of lemon and kiwifruit and the fruit liqueur during freezing and thawing of the fruit liqueur. It is generally said that the freezing point of food is about - 2 C to - 5 C, and at further lower temperature, freezing of food causes it to become frozen (Kato, 1966). As shown. in Fig. 1, each freezing point of lemon and kiwifruit ac- quired in this experimental condition was lower than the temperature they had said. The sarcocarp of lemon would require 2.8 h (165 minutes) and the sarcocarp of kiwifruit would require 3.4 h (205 minutes) to reach the general freezing point of - 2 C. Moreover, the sarcocarp of lemon required 3.5 h (210 minutes) and that of kiwifruit required 4 h (240 minutes) to reach the thawing temperature. This difference was thought to depend on the size of the samples as kiwifruit requires longer freezing and thawing time than a lemon. Generally, - 25 C is the freezing point for liquor with 35% alcohol. Therefore, the liquor did not freeze because the temperature of the freezer of this study was - 20 C. 2. Ascorbic acid, polyphenols, total acidity and color difference in the fruit liqueur with and without sugar At first, to clarify the influence of freezing pretreatment on the dissolution of components from fruits to the liquor, the contents of ascorbic acid, polyphenol, and the titratable acidity as an index of the organic acid content in the fruit liqueur without sugar were measured. Fig. 2 shows the VC content of the fruit liqueur. The amount of VC in the freezing pretreatment sample was almost the same as that in the no-freezing pretreatment sample after 4 days of soaking time, suggesting treatment that the freezing had no influence on the amount of VC in the liqueur. In the "0 soaking day" sample, one day after soaking, a difference in VC dissolution was not seen between the freezing pretreatment and no-freezing pretreatment. The freezing pretreatment sample was soaked in the li- quor at - 20 C before soaking at 20 C, and at this time, VC in the fruits was already partly dissolved in the li- quor. That is why there was a difference in VC content between the freezing pretreatment and the no-freezing pretreatment samples before the start of soaking at 20 C (0 soaking time). The dissolution of VC from the fruits to the liquor depended on the time in which the fruits were soaked in the liquor and not directly on the soaking time with freezing pretreatment. The AsA content of the fresh lemon and kiwifruit measured was 38.7 and 60.5 mg/100 g, respectively; on the other hand, the DHA content in the fresh fruits was zero. Moreover, the amount of the fruit liqueur extracted from 300 g of fruits was in the range of 600 ± 19 ml. Theoretically, if all the VC of the lemon and kiwifruit dissolved in the liqueur, the yield would be 19.35 mg/ 100 ml and Fig. 1 Changes in the temperature of freezing and thawing of fruits at 20 C in the process of liqueur making, sarcocarp of lemon ;, sarcocarp of kiwifruit ;, liqueur 46 (268)
Fig. 2 VC contents of fruit liqueur during soaking VC : a, freezing pretreatment') (total VC) ; 0--, no freezing pretreatment2) (total VC) ; -A--, freezing pretreatment (AsA), --A--, no freezing pretreatment (AsA) ; U, freezing pretreatment (DHA) ; --0--, no freezing pretreatment (DHA) 1) Sample was frozen with the white liquor (Japanese distilled spirits) at - 20t for 24 h and allowed to stand at 20t for 0-4 days. The soaking time 0 means that there is no standing time at 20t after the sample was frozen at - 20t for 24 h. 2) Sample was soaked in the white liquor (Japanese distilled spirits) and left to stand at 20 C for 0-4 days. 30.25 mg/100 ml. In this experiment, the actual percentage of the amounts of VC dissolved in the fruit liqueur from the fresh fruits of lemon and kiwifruit at a 2-day soaking time was 74 and 77%, respectively. In the case of kiwifruit liqueur, the percentage of VC in both the frozen pretreatment and the no-freezing pretreatment samples increased from 77% to about 90% at a soaking time of 4 days compared to lemon which showed no change at the same soaking time. It is suggested that most of the ascorbic acid in the fresh fruit was extracted into the fruit liqueur during the second and fourth day of soaking. The amount of VC decreased to almost 0 when the samples were soaked at room temperature for 90 days (data not shown), and it was clarified that the remarkable decrease in VC in the liqueur was due to the long-term soaking of the fruits, though it was clear that VC still remained in the liqueur after 4 days soaking. Fig. 3 shows the change in the polyphenol content in the fruit liqueur with soaking time. After the first day of soaking, no significant difference in the amount of polyphenol between the freezing pretreatment and the nofreezing pretreatment was observed. The polyphenol content of the fresh lemon or kiwifruit was 133 and Fig. 3 Changes in the polyphenol contents of fruit liqueur during soaking Polyphenol :, freezing pretreatment' ; --0--, no freezing pretreatment2) 1) 2) Samples are the same as those in Fig. 2. (269) 47
J. Cookery Sci. Jpn,. Vol. 40 No. 4 (2007) 72 mg/ 100 g, respectively. If all the polyphenol dissolved in the liqueur, the yield would be 66.5 mg/100 ml and 36.0 mg / 100 mi, respectively. The percentage of the amount of polyphenol dissolved in the fruit liqueur from the fresh lemon and kiwifruit was about 40% and 70%, respectively, at 4 days of soaking time. Moreover, it was clear that the amount of polyphenol in lemon and kiwifruit slightly increased after 4 days and continued to increase at 90 days soaking, finally reaching 29.1 ± 1.2 mg/ 100 ml and 22.7 ± 0.6 mg/ 100 ml, respectively. It is obviously seen that polyphenols were much more stable than ascorbic acid in the fruit liqueur and remained over a long period of time. It is thought that the activity of polyphenoloxidase remained low in 35 % of alcohol solution. Next, to determine the amount of dissolved organic acid in the fruit liqueur, the titratable acidity was measured. Fig. 4 shows the change in the titratable acidity of fruit liqueur with soaking time. The titratable acidity of fresh lemon and kiwifruit was 5.01% and 0.96% as citric acid, respectively. As shown in this figure, the titratable acidity of fruit liqueur in lemon increased due to the freezing pretreatment, and a significant difference was observed between the freezing pretreatment and the nofreezing pretreatment, but no difference was seen in the kiwifruit. In the case of lemon, the acidity of which was 1.90 ± 0.03%, obviously increased at room temperature in a soaking time of 90 days. This is probably because lemon is originally rich in citric acid. These results indicate that the effect of freezing pretreatment on the amounts of dissolved components in the fruit liqueur was small, and it was clarified that the influence depends chiefly on the soaking time in the liquor. Fig. 5 shows the change in the color difference between the white liquor and the fruit liqueur as a function of time. There was little difference in the color value between the freezing pretreatment and no treatment up to 4 days of soaking. This suggests that a short term soaking does not cause any change in the color of the liqueur. Generally, it is not possible to determine the significance of difference in the case of LIE 3 = 3 or less. On the other hand, the Z1E value of no-treatment was 5.6 ± 0.3 and 8.5 ± 0.3, respectively, lemon and kiwifruit showing a drastic increase at a soaking time of 90 days (data not shown). It is thought that the dissolved components gradually reacted with other substances and changed the color of fruit liqueur during soaking. Table 1 shows the relative ratio of the content of fruit components in the liqueur by freezing pretreatment soaked with sugar and without sugar. In all the values measured, the relative ratio was less than 1.0. This shows that the dissolution of components fruits into the liquor is slightly suppressed from freezing by the addition of sugar. It was thought that the cell membranes the fruits were affected by the addition of sugar to the fruit liqueur. As for lemon and kiwifruit, the difference in the amount of dissolved ascorbic acid or polyphenol was very small between the freezing pretreatment samples and the no-pretreatment ones. There appeared to be no effect of freezing on the dissolution of components alcohol soaked samples. This suggests of in the that the amounts of dissolved components were more greatly influenced by Fig. 4 Changes in the titratable acidity of fruit liqueur during soaking Titratable acidity : 0, freezing pretreatment1 ; --0--, no freezing pretreatment') 1) 2) Samples are the same as those in Fig. 2. 48 (270)
Fig. 5 Changes in the color difference of fruit liqueur during soaking Color difference, freezing pretreatment' ; --0--, no freezing pretreatment2) 1) 2) Samples are the same as those in Fig. 2. Table 1. Effect of addition of sugar to freezing pretreatment fruit liqueur on the dissolution of fruit components') 1) Data represent the relative ratio of the value of the sugar-added sample to that of the no-sugar-added sample. Without sugar was considered as 1 2) The amounts of DHA are zero for both with and without sugar added, when the soaking time is 0 day. the conformational change in the cell membranes of fruits due to the infiltration of alcohol, than by the freezing pretreatment. The damage to the plasma membranes of agricultural products caused by the freezing and thawing could be measured by electrical and rheological properties (Ohnishi et al., 2002). The influence of freezing pretreatment, alcohol soaking and sugar addition on the function of the cell structure 3. Impedance analysis was analyzed by measuring the impedance. It is generally known that, when the structure of the cell membranes of vegetables and fruits changes and loses its function, the impedance in the low frequency area decreases, and the circular arc of the Cole-Cole plot obtained from the impedance disappears (Cole, 1932). In this study, the influence of alcohol, freezing pretreatment and sugar on the cell structure as a model was compared by the impedance of the samples. At first, the effect of freezing using apple and ethanol and addition of sugar when the apple was soaked in 35% of ethanol as a model of fruit liqueur was examined. As shown in Fig. 6, soaking in alcohol greatly decreased the impedance in the low frequency area, and freezing decreased the impedance further compared with the fresh sample. However, the circular arc in the Cole-Cole plot almost disappeared the same level both in the freezing pretreatment at and the alcohol treatment, and it was suggested that the struc- (271) 49
J. Cookery Sci. Jpn. Vol. 40 No. 4 (2007) Fig. 6 Effect of freezing treatment on the impedance and Cole-Cole plot of apple tissue soaked in 35% alcohol solution with added sugar *, fresh apple ; 0, freezing pretreatment at 20t ; -0-, no freezing pretreatment (only alcohol soaking) ;, 25 % sugar added and freezing pretreatment at - 20t A, 25 % sugar added with no freezing pretreatment at 20 C. Samples except for fresh apple were soaked in 35% alcohol solution. The ratio of sugar added is the same as in Table 1. Fig. 7 Cole-Cole plot of apple tissue in 35% alcohol solutions at various soaking times *, fresh apple ; 0, 0 h (immediately after soaking) ; 0, 1 h ; 0, 2 h ; A, 3 h ture of the cells changed and their function substantially decreased. On the other hand, when sugar was added to the alcohol-treated sample without freezing, the impedance was found to be obviously larger than that of the fresh samples, and the Cole-Cole plot was also clearly observed, indicating that sugar was effective in protecting the cell membrane. It was reported that a Cole-Cole plot was observed when potato was soaked in mannitol solution at a concentration of 0.7 M (Dejmek and Miyawaki, 2002). In this study, the concentration of saccharose used in the actual experiment corresponded to 0.58 M. However, when sugar was added to the freezing pretreated samples, the impedance decreased and the Cole-Cole plot was not observed. As a result, any additive effect of freezing was not found because the semipermeability of the cell structure was lost only by soaking in 35% alcohol that corresponds to the fruit liqueur, and it was possible for sugar to inhibit the loss of function of the membranes caused by alcohol. It was indicated that the freezing pretreatment decreased the effect of sugar on the protection of the cell structure. As shown in Fig. 7, the circular arc of the Cole-Cole plot has become small with the increasing soaking time, and it completely disappeared in 3 h. It was presumed that the structure of the cell membranes changed followed by the disappearance of their function starting at about 2 h after soaking and was completely damaged at 50 (272)
3 h after soaking. The disappearance of the Cole-Cole plot suggests a serious change in the cell structure (Ohnishi et al., 2004). The dissolution of components of a 1 cubic cm sample is suggested to occur after more than 3 h of alcohol soaking. The measurement of impedance was carried out using apple in this experiment, and this result could be applied to other plant foods, because the cell membranes have many common characteristics. of plants including lemon and kiwifruit It was clarified that the effect of combining freezing pretreatment and alcohol soaking was hardly observed in the dissolution of components such as VC and polyphenol in the preparation of fruit liqueur compared to alcohol soaking only. These results suggested that the effect of freezing treatment on the cell structure relative to the dissolution of the components were not seen because the influence of alcohol itself was so large. It was also found that about 70% of VC in the fruits remained in the liqueur after soaking for 4 days. We acknowledge Prof. Osato Miyawaki (Ishikawa Prefectural University) REFERENCES for the measuring of impedance. Cole, K. S. (1932), Electric Phase Angle of Cell Membranes, J. Gen. Physiol., 15, 641-649 Dejmek, P. and Miyawaki, 0. (2002), Relationship between the Electrical and Rheological Properties of Potato Tuber Tissue after Various Forms of Processing, Biosci. Biotechnol. Biochem., 66, 1218-1223 Fujimaki, A. (2001), Fruit Wine: Flower Wine and Medicinal Wine, Graph Co., Tokyo, pp. 12-13 Ishida, Y. (1993), Measurements of Vegetable Colors, Science of Cookery, 26, 378-384 Kato, S. (1966), Syokuhinreitou no Riron to Ouyou, Kourin Syoin, Tokyo, pp. 321-323 Mazza, G., Fukumoto, L., Delaquis, P., Girard, B. andewert, B. (1999), Anthocyanins, Phenolics, and Color of Cabernet Franc, Merlot, and Pinot Noir Wines from British Columbia, I Agric. Food Chem., 47, 4009-4017 Murakami, T., Kasai, M. and Hatae, K. (2002 a) Effect of Soaking and Freezing before Cooking on the Softening Rate and Cooking Time of Azuki Beans, I Home Econ. Jpn., 53, 887-892 Murakami, T., Kasai, M. and Hatae, K. (2002 b) Effect of Soaking and Freezing before Cooking on the Quality of Sweetened Azuki Paste, I Home Econ. Jpn., 53, 893-899 Ohnishi, S., Fujii, T. and Miyawaki, 0. (2002), Electrical and Rheological Analysis of Freezing Injury of Agricultural Products, International Journal of Food Properties, 5, 317-332 Ohnishi, S., Shimiya, Y., Kumagai, H. and Miyawaki, 0. (2004), Effect of Freezing on Electrical and Rheological Properties of Food Materials, Food Sci. Technol. Res., 10, 453-459 Soufleros, E. H., Irini Pissa, Petridis, D., Lygerakis, M., Mermelas, K., Boukouvalas, G. and Tsimitakis, E. (2001), Instrumental analysis of volatile and other compounds of Greek Kiwi wine; sensory evaluation and optimisation of its composition, Food Chemistry, 75, 487-500 Tominaga, A., Mizukami, K. and Arikawa, T. (2001), Changes in Free Amino Acids, Sugars, Organic Acids and Browning of Umeshu (Japanese Plum Liqueur) During Long-Term Storage, J. Home Econ. Jpn., 52, 1133-1138 Yasui, Y. andhayashi, M. (1993), KouenYousisyu, J Soc. Anal. Chem., 42, 578 (Received Sep, 27, 2006 Accepted May, 7, 2007) (273) 51
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