Koki Yokotsuka

Comparison of soluble proteins in juice and wine from Koshu grapes

Abstract Proteins were separated from Koshu grape juice and wine by precipitation with ammonium sulfate. The protein fractions were further fractionated by gel electrophoresis and gel isoelectric focusing, followed by amino acid analyses. The juice contained more than eleven protein fractions with molecular weights between 13,000 and 65,000, and their isoelectric points were between 3.6 and 10.5. The wine also contained more than eleven protein fractions with molecular weights between 21,000 and 65,000, while their isoelectric points were between 3.6 and 11.0. All the juice proteins and some major wine proteins were glycoproteins. The same three protein fractions were present in both juice and wine. The other juice proteins were lost during wine-making and thus, were not detected in the wine. About half of the proteins detected in the wine were not observed in the juice. Some juice proteins were bound to the flavonoid phenolics extracted from the wine and were removed as insoluble precipitates. There was specific interaction between wine flavonoids and juice proteins.

Characterization of thermostable invertase from wine grapes

Abstract The levels of invertase activity were found to vary significantly among 14 varieties of grapes tested. The crude invertases were, however, similar in both pH and temperature optima, as well as pH and thermal stabilities. The enzymes showed a high degree of thermostability and were also stable at acidic pH and high concentration of alcohol. A significant level of invertase activity persisted in several white table wines. The optimum activity of the enzyme purified from Semillon grape was observed at about 75°C and it was stable up to 70°C. Although the enzyme was stable between the pH 2 and 8, the optimum pH for its activity was about 4. The enzyme, whose molecular weight was estimated to be 65,000 by SDS polyacrylamide slab gel electrophoresis, was found to be a glycoprotein with a total carbohydrate content of 33%. This enzyme showed activity toward sucrose and raffinose, but was inactive on the other disaccharides tested.

Purification and properties of invertase from Muscat Bailey A grapes

Abstract A grape invertase was purified from Muscat Bailey A juice by salting out with ammonium sulfate and successive chromatographies on Sephadex G-100 and Con A-agarose to a homogeneous state as confirmed by polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was 72 kDa in gel filtration chromatography. However, SDS polyacrylamide gel electrophoresis revealed three bands with molecular weights of 56 kDa, 25 kDa and 24 kDa. Affinity staining of Western blots with lectins indicated that the enzyme was a glycoprotein. The optimum pH for the enzyme reaction was 3.5 and the optimum temperature 80°C. The enzyme was stable from pH 2.7 to 6.4, and up to 80°C. The transfructosylation reaction could not be observed. The K m value of this enzyme for sucrose was 4.4 mM at pH 4.0, but as the pH of the reaction mixture increased, the K m value decreased sharply. From the dependence of the V max and K m values on pH, the ionization constant (p K e ) of one of the two essential ionizable groups of the free enzyme was determined to be 2.7, suggesting that this essential ionizable group was a carboxyl.

PURIFICATION AND SOME PROPERTIES OF THERMOSTABLE INVERTASE FROM WINE

Abstract Invertase from a white table wine made from Semillon grapes was purified to homogeneity on polyacrylamide gel electrophoresis. The enzymatic and physicochemical properties of the enzyme were compared with those of invertase purified from Semillon grape juice. The invertases from the two sources showed similar properties, suggesting that the wine invertase originated from the juice and was stable during the vinification and aging processes.

Extraction of anthocyanins from muscat bailey A grape skins

Abstract Anthocyanins from the skins of Muscat Bailey A grapes were extracted at different temperature in the presence of various concentrations of SO 2 . Diluted wine spirits containing 5 to 25% ethanol were used for the extraction. The extracts were separated by two-dimensional paper chromatography and were found to contain the following pigments, in a decreasing order of concentration, malvidin-3-monoglucoside, malvidin-3, 5-diglucoside acylated with p -coumaric acid, peonidin-3, 5-diglucoside, malvidin-3, 5-diglucoside, malvidin-3-monoglucoside acylated with p -coumaric acid, petunidin-3-monoglucoside, delphinidin-3-monoglucoside, and peonidin-3-monoglucoside. The red color of the extracts was mainly due to the extracted free anthocyanins. A very small amount of polymeric anthocyanins was formed during the extraction. The total phenol, and total and individual anthocyanin contents of the extract increased with increasing temperature as well as increasing SO 2 and ethanol concentrations. The rates of extraction of these anthocyanins varied with the kinds and amounts of the individual anthocyanins and the extraction conditions.

Polyphenoloxidase from six mature grape varieties and their activities towards various phenols

Abstract Juices were prepared from three white and three red grape varieties harvested at full maturity and comparative studies on their oxygen-uptake, absorbance at 420 nm (degree of browning), polyphenoloxidase (EC 1.10.3.1; PPO) activity, and their phenol compositions were done. There was no correlation among the amounts of oxygen-uptake and oxidizable phenols in the juices and their degree of browning. However, there was similarity among the PPO from the six grape varieties in their general enzymatic properties and substrate specificity towards twenty-five phenols. A partially purified PPO fraction from Koshu juice, which did not contain free phenols, showed strong activity towards (+)-catechin, (−)-epicatechin, caffeic acid, catechol, pyrogallol, and protocatechuic acid (oxidizable phenols), but had no activity towards the other fifiteen phenols. The oxidizable substrates were not always the only limiting factor in the oxidation and browning of phenols by the PPO. Some unoxidizable phenols such as gallic acid, p-cresol, and tannic acid which were not substrates for PPO inhibited the oxidation of the oxidizable phenols except pyrogallol which was not inhibited by gallic acid. On the other hand, hydroquinone promoted the oxidation of the oxidizable phenols except protocatechuic acid. These showed that there were competitive reactions and synergism during the enzymatic oxidation of phenols.

Removal of red blush and bitterness from white wine by partial hyperoxidation of juice from the pink-skinned koshu variety

Abstract White wine which is produced using sulfited juice from koshu grapes, the most important cultivar native to Japan, sometimes has bitterness and/or astringency and a red blush. Blowing compressed air through unsulfited juice for a short period of time results in less bitterness and/or astringency and no red blush, thereby improving wine quality, whereas conventional hyperoxidation produces thin, watery wine with little colour and poor body. It was found that the bitterness and/or astringency of wine made from sulfited juice is due to oligomeric and polymeric tannins extracted from skin and perhaps seed of soft koshu grape berries during crushing, stemming, and pressing, whereas the red blush is probably due to excess caffeic acid derivatives, anthocyanins or anthocyanin–flavonoid complexes, and cyanidins produced from procyanidins. Partial hyperoxidation removed the phenols responsible for the bitterness and/or astringency and the red blush via enzymatic oxidation (uninhibited by SO2), polymerisation, and insolubilisation.

Changes in Amount of Nitrogenous Compounds from Skins and Seeds of Four Grape Cultivars During Extraction Using Juice- or Fermenting Must-Like Model Solutions

Changes in the amounts of nitrogenous compounds (total and free amino acids, peptides, and proteins) were investigated during extraction under oenological winemaking conditions from seeds and skins of two Japanese cultivars (Koshu and Muscat Bailey A) and two European cultivars (Chardonnay and Cabernet Sauvignon) using juice-like model solutions and fermenting must-like model solutions. Two maceration systems, one prior to fermentation (without ethanol) and the other, during fermentation (with ethanol), were used. The amounts of nitrogenous compounds extracted increased with time. The amounts extracted varied with the cultivar. The nitrogenous compounds were gradually extracted from seeds, whereas those from skins were almost completely extracted on the first day. The presence of nitrogenous compounds, in particular free amino acids extracted from seeds and/or skins, may be relevant as nutrients for yeast growth during fermentation.