| It is not seen in literature how the color of pigment in chestnut (Castanea mollissima Blume) change when chestnuts occur to brown in processing. In our experiments, fresh chestnuts were dried at 45℃for 12h, with shells decorticated and coats removed, and the kernels were crushed and extracted pigment from chestnut kernels with ethanol, acetone, ether, chloroform, and petroleum respectively. All the crude extracts were scanned through an ultraviolet spectrometer in the range of 200 - 700 nm. The highest absorbing wavelength was shown at 441 nm, under which all the crude extracts were measured for efficiency of different solvents, and the results demonstrated that ether was the best extracting solvent. Then an orthogonal design (L934) was adopted and corresponding experiments were conducted among such factors as the ratio between materials and solvent (R), duration (D), temperature (T) and pH for the best extracting conditions, and the results suggested the following: R = 1:3, D = 1.5 h, T = 35℃, and pH = 7. Finally the content of crude pigments in chestnut kernels was indicated at 2.8% by the experiments. The distribution of pigment in outer chestnut are 2 times as much as the inner ones by examining ABS of extracting solution from same mass which sliced chestnut from the diameter of two and third.The yellow pigments in chestnut kernels were fat-soluble, dissolvable in organic solvents but not in water. Crude extracts were yellow in color, but brownish yellow if thickened. Their absorbing wavelength was chiefly at 416, 441, and 470nm in visible light, and 240 and 296nm in UV; they also exhibited a weak fluorescence at 310 and 340 nm. Lights faded the pigments; temperature showed no apparent effect on stability of the pigments, but affected remarkably when exceeding 100℃. The acidity in terms of pH from 1 to 14 made the color turn from brownish yellow to orange, and to green in extremely acid solutions. Metal ions such as Cu2+,Fe3+,Ca2+,Al2+,Sn2+,Mn2+,Zn2+ had no significant effects on the pigments, but Pb2+ did. Food additives such as citric acid, sucrose, and Vc influenced the stability of the pigments as their concentrations increased. Both sodium sulfite and sodium chloride stabilized the pigments with their density increment. The treatment with microwave and ultrasonic wave displayed some destruction on the pigments. The extract of the browned chestnuts absorbed more lights than the normal one, meaning that the browning of the chestnut kernels was not due to the yellow pigments of the kernels themselves but to other materials that masked the normal yellow color.The crude extracts of chestnut kernels were isolated via a column chromatography filled with silica gel and alumina (3:1, M/M), and washed sequentially with n-hexane, dichloromethane, and methanol to obtain non-polar, weak-polar, and polar fractions, and the polar fraction was then methylated. All the fractions, including the methylated one, were subject to a gas chromatography-mass spectrometry (GC-MS) analysis, and a total of 68 compounds were found. There were 19 compounds in the non-polar fraction, mainly Alkanes and Olefins, among which Heptacosane accounted for 29.937%, Hentriacontene for 23.945%, and Nonacosene for 22.196%. Squalene, one of the 19 compounds in the non-polar fraction, contains conjugated double bonds composed of six units of isoprene that belong to triterpene compounds with chromophores; whereas the others have no this kind of chromophore. Weak-polar fraction consisted of 29 compounds, mostly aldehydes and esters, among which the relative amounts of Linoleic acid ethyl ester, 9-Octadecenoic acid, and Ethyl Oleate were 19.216%, 16.406%, and 11.976% respectively. Conjugated linoleic acids and esters themselves are colorless, but they can show colors when combined with other pigments. Meanwhile, functional ingredients of Linoleic acids, Stigmastans,β-Tocopherols and Flavonoids were also found. Inside polar fraction there were 20 compounds found, mainly organic acids, among which were Nonanoic acid (25.514%), Hexadecanoic acid (15.2833%), Octanoic acid (14.8311%), etc. These kinds of compounds do not have chromophore. Carotenoids and other pigment compounds were not found by GC-MS.High-speed Countercurrent Chromatography (HSCCC) was used to isolate the yellow pigments, with the mixed solvents of n-hexane, ethyl acetate, ethanol, and water (6:1:6:1, V/V) as the washing reagent. Three main peaks appeared, accounting for 64.62%, 19.07%, and 13.91% respectively. When examined under UV, the first peak was colorless,λmax :220 nm; the second, yellow,λmax = 444 nm, 473nm,220nm; and the third, colorless,λmax :296 nm; the fixed phase with the remaining solvent mixtures showed a yellow color,λmax: 423 nm ,446 nm,471 nm.,220 nm,296 nm。A silica thin layer chromatography, with spreading solvents of n-hexane and ether (1:2.5, V/V), was employed to examine the components of both the crude extracts and the fractions isolated by HSCCC. Two yellow spots were obtained, with R?1 = 0.38,and R?2 = 0.96. HPLC analysis indicated that the yellow pigments in chestnut kernels containedβ-carotene and lutein; the second peak of HSCCC, R?1, were lutein; the fixed phase with the remaining solvent mixtures and R?2 wereβ-carotene. The content of lutein in fresh chestnuts is 0.42mg/100g,β-carotene`s contents is 0.16mg/100g.Therefore, the yellow pigments in chestnut kernels are composed ofβ-carotene and lutein by analyzing with UV,TLC,GC-MS, and, HPLC.
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