Evaluation of cysteine as anti-browning substance of fruit

It had been demonstrated by Fujii that poisonous nature of tetrodotoxin is counteracted by the action of SP (sodium sulfite solution adjusted to pH 5.5 with ortho phosphoric acid). According to him, the antidotal virtue of SP is based on the change of chemical configuration of the toxin due to the reduction of OH groups in the molecule and the addition of sulfite to it. If it is the case, SP might exert similar influence on the several phenomena in which polyphenol compounds take an essential part such as browning of apple or potato and astringent taste of kaki (Japanese persimon). The former is brought about by the action of phenol oxidase on the natural polyphenol substance, precursor of colored material such as chlorogenic acid or catechin, and astringency of kaki is attributed to soluble tannin in the fruit. If these polyphenol compounds are chemically modified by SP as tetrodotoxin, browning of apple shall be inhibited and astringency of kaki shall be removed. Indeed, both presumed efficiencies of SP were confirmed and invsetigated as reported in our foregoing papers. However, SP must be carefully employed, because it is not ideal but sometimes harmful as food additions. Recently, Fujii discovered that cysteine also counteracts with tetrodotoxin under certain condition during the course for survey of better antidotal substances on the basis of his opinion. According to his idea, it must interfere with browning of apple as SP. In addition, employment of cysteine is desirable, from the point of enrichment of amino acid too. Therefore, cysteine was evaluated as to anti-browning efficiency. Pieces of rinded apple were immersed into 5×10^-3M cysteine solution of various pHs for 30 minutes at room temperature and the color of their surface was observed at 5 and 30 minutes, 1, 2, 20, 40 and 90 hours after immersion. It was established that browning of apple was clearly retarded by cysteine, especially at pH over 6. On the other hand, although browning of apple had been more or less retarded, extinguishing peculiar pink color appeared one day after immersion into cysteine solution of weak acid, and distinctive pink one after a few days which clearly differ from brown color of non-treated apple. Similar efficiency of cysteine was also established with apple juice. A piece of rinded apple was quickly homogenized into a double volume of cysteine solution with a Waring blender and filtered by suction. Optical density of the juice prepared was estimated by our usual procedure using an electric colorimeter with blue filter at 1.5 and 20 hours after preparation. Incidentally browning of the juice was qualitatively observed. When water or buffer of pH 6 was employed as solvent in stead of cysteine solution, the juice became soon dark brown and high optical density was determined as usual. On the other hand, lower values were estimated, as concentration of cysteine increased, and no browning was observed when 5×10^-4 or 10^-3 M cysteine solution had been employed. However, pinkish color appeared in the case of lower concentration at 1.0^-4 M or below, similar to treatment with cysteine at weak acid. Since apple became brown at once after rinderin g or homogenizing, it is difficult to make uniform examination with flesh one. Rinded apple was dehydrated by homogenizing it into cold acetone. The procedure was repeated and light white powder was obtained after drying in a desicator in vacuo. An extract of the acetone powder became brown soon, when chlorogenic acid or pyrogallol was added. However, coloration of the mixture was not observed if the boiled extract had been employed. The browning was inhibited by SP and cysteine as flesh apple and sodium sulfate had no effect. Using this model system of the extract of acetone powder and pyrogallol, inhibition of browning by SP and cysteine of various concentrations could be uniformely investigated. By comparing the efficiencies of both inhibitors, it was observed that cysteine has anti-browning virtue of similar level to that of SP. When apple had been immersed into cysteine solution for a longer period, gas with indisposed odor was formed and the solution became turbid white. Gas was bubbled through lead acetate solution and black precipitates contaminated with white one were produced. Precipitates were gathered by filtration and washed a few times with 2 N sodium hydroxide solution which dissolved the white precipitate but not the black one. The black precipitate was then dissolved in 2 N nitric acid and the solution was neutralized with sodium hydroxide. Through this solution, hydrogen sulfide gas was bubbled and black precipitate was again formed, which indicated that the precipitate is lead sulfide and gas formed contains hydrogen sulfide. Into sodium hydroxide washing of the first precipitates, sulfuric acid was added and white precipitate was produced. When hydrogen sulfide was passed, black precipitate was also formed. These showed that the washing contains at least lead derived from the first white precipitate. Thus, at any rate, indisposed odor of gas must be attributed to hydrogen sulfide. However, formation of hydrogen sulfide did not be observed under the same condition when apple had been omitted. Similarly, if the apple was immersed into cysteine solution and warmed at 60℃ for 5 or 10 minutes, formation of gas also prohibited. These observations suggested that formation of gas, hydrogen sulfide, is enzymatic reaction and the enzyme concerned may be cysteine desulfhydrase. However, apple became brownish pink in color when it had been warmed for 10 minutes, although no coloration as well as gas formation was seen in the case of 5 minutes' heating. White precipitate formed after longer immersion was obtained by centrifugation at 8,000 r.p.m. for 30 minutes, washed with water, alcohol and ether and dried in vacuo. Infrared spectrum of the precipitate was estimated and established to be different from chlorogenic acid, cysteine and cystine, but to be similar to precipitates formed from rinded astringent kaki by the same treatment with SP or cysteine. Since cysteine is thus useful in enrichment of amino acid as well as in prohibition of browning, effects of cystine and methionine on browning of apple were investigated. It was found that cystine has no effect and browning of apple was exceedingly accelerated by methionine. Trichromatic coefficients of colors of control brown, pink with cysteine of weak acid, accelerated dark brown with methionine and inhibited browning with SP were estimated. By consulting these data on the map of CIE system, dominant wave lengths of 577, 588, 582 and 574 were estimated. According to Fujii's idea, cysteine should remove astringency of kaki on the same mechanism. Rinded kaki was immersed into 2×10^-2M SP or 2×10^-3M cysteine solution for 3 days and white precipitates were obtained in both solutions. They were dehydrated by washings with water, alcohol and ether and their infrared spectra were determined. It was found that spectra were quite resemble in each other, suggesting that tannin of kaki became insoluble by treatment with SP as well as cysteine and that astringency was removed. Therefore, it may be presumed that the mechanism of cysteine is similar to that of SP.