Fumio Nagayama

STUDIES ON THE BROWNING OF FISH FLESH-IV

Ultraviolet absorption spectra, fluorescence, and some characteristics on paperchromatography were observed on sugar solution in 0.1M phosphate buffer, pH 6.8 (0.05M) after autoclaving for one hour at 115° in the presence of amino acid 0.05M) Among four amino acids (arginine, lysine, histidine and glycine), lysine shows the strongest accelerating effect for production of brown color and ultraviolet absorption (Table 1). Ultraviolet absorption maximum is found in the region between 268 to 295mμ (Figs. 2 and 3), and the spectra were considered similar to that of reductone rather than furfural. By paperchromatography using n-butanol-acetic acid-water, (4:1:2), the following points were investigated: 1) By ninhydrin, no spot is detected other than amino acid. 2) Amino acid and sugar produce fluorescence without loss of their characteristics as Rf and reactivity for spot detection. 3) Most of brown color is not developed, while fluorescence is moved as same as sugar and amino acid. 4) Inorganic ortho phosphate does not combine with sugar, amino acid, or other reaction products.

STUDIES ON THE BROWNING OF FISH FLESH-IX

In this experiment, it was tried to fractionate and characterize the browning products in glucose-lysine system. Colored products and fluorescent products were cationic. Brown pigments were combined irreversibly with Dowex 50, and they showed fluorescence. Colorless fluorescent substances having ultraviolet absorption at 250 mμ were able to be eluted from Dowex 50 with dilute HCl. On paper chromatograms, 4-5 colored, 5-6 fluorescent, and 5-6 ninhydrin reactive spots were detected. Spots corresponding to lysine, glucose, and glucosamine displayed strong fluorescence. A fluorescent and ninhydrin reactive spot with smaller Rf value than lysine was detected. Brown pigment which could not be developed showed considerable fluorescence. All of fractions by paper chromatography showed maximum fluorescence at 450 mμ, and maximum absorption at 265-275 mμ.

Studies of β-Galactosidase of the Rainbow Trout Liver-III

The activities of β-galactosidase and lactase, and the pattern of β-galactosidase isozymes were investigated to see the effect of dietary lactose and starch on the enzyme of the rainbow trout. The lactase activity of the fish tissue was increased by the high carbohydrate feeds, especially by the high lactose feed which was reported to cause the elevation of the lactase activity in the calf intestine2). On the other hand, the β-galactosidase activity in the liver and kidney was rather decreased by the high carbohydrate feeds, although the β-galactosidase of E. coli was known to be induced by lactose in the medium3). These results may be a possible evidence for the concept that the animal β-galactosidase (o-NPGa-ase) is not identical with lactase4-8). Three isozymes of -galactosidase (I, III, and IV) were isolated by a Sephadex G-200 column from all tissues of the rainbow trout yearling, but the isozyme II being found in the 2-year fish1) was not detected. The pattern of the isozyme seemed to be tissue specific, and not influenced by the dietary composition. The molecular weights of the isozymes were the multiples of 57, 000: ×6 (I), ×3 (II), ×2 (III), and ×1 (IV). The lactase activity was not recovered from a Sephadex G-200 column.

STUDIES ON THE BROWNING OF FISH FLESH-VIII

The browning reaction of glucose-lysine system was studied to explain the accelerating mechanism of the reacting by phosphate or borate. Intensities of color, fluorescence and ultraviolet absorption of the system increased in parallel with each other. Decrease of fluorescence did not occur even though the browning reaction was extended. The fluorescence of the system was supposed to be due to not only precursor of brown pigment but final products of the reaction. Ultraviolet absorption spectrum of the system containing phosphate differed from the one containing borate. Borate induced the loss of the reducing power of glucose in the absence of lysine. It was considered that phosphate may accelerate the browning reaction by its simple catalytic action. On the contrary, borate may possibly accelerate the reaction through the formation of its complex with sugar. One of the possible way of the reaction induced by borate is aldehyde formation by opening of pyranose ring, and another way is ketose (sorbose) formation.

STUDIES ON THE BROWNING OF FISH FLESH-VII

Fluorescent spectrum and flavor were investigated on the sugar-amino acid systems in 0.1M phosphate buffer, pH 6.5, which were autoclaved for 90 minutes at 113°C. Fluorescence of the system consisted of light of shorter wavelengths than 620mμ and showed the maximum intensity at ca. 480mμ. These were similar to that of unsaturated (hetoro) cyclic nitrogenous compounds which are commonly used as fluorescent whitening agents. Amino acids having unsaturated ring, tryptophan, phenylalaine, and histidine, produced strong fluorescence by the reaction with sugar. Lysine and arginine which yield intense color and fluorescence were also supposed to form unsaturated compounds. On the other hand, imino acids with saturated ring formed little color and fluorescence. Neither color nor fluorescence was produced with aspartic and glutamic acids. The sugar-amino acid systems produced flavors by heat in parallel with their browning. The produced flavors were generally cocsidered to be underiable for foods with a few exception.

STUDIES ON THE BROWNING OF FISH FLESH-VI

Glucose and ribose cause browning in the presence of amino acid, and the browning reaction seems to be accelerated by inorganic phosphate. In this experiment, some glycolytic intermediates, hexose-phosphates and adenosine phosphates, were autoclaved in the presence of amino acid. G6P, F6P and FDP produced brown color, but GIP, AMP and ATP did not produce any visible color. In general, sugar phosphates and adenosine phosphates liberated inorganic phosphate and lost sugar during autoclaving. However, the degrees of phosphate-liberation and loss of sugar were greater in G6P, F6P and FDP which produced brown color than in others. Adenosine phosphates which contain ribose caused no browning, although it liberated inorganic phosphate and lost ribose by autoclaving. It is considered that the browning of hexose phosphates except G1P is introduced by the liberation of inorganic phosphate from sugar, being followed by the sugar-amino reaction accelerated by the catalytic action of inorganic phosphate. Thus, the possibility that hexose phosphates may be involved in some extent in the browning of fish flesh by heat must be considered, although their contents are lower than these of free sugars in fish flesh.

Studies on the Lipoxidase in Fish Tissues-III

That the loss of vitamin A content during the storage of fish liver was markedly affected by the temperature and seemed to be caused by a catalytic action of a certain enzyme system was previously reported by the present authors1). The authors, now, observed the enzymatic action of the extract of fish liver in the oxidation of vitamin A, and attempted to determine the identity of the soybean lipoxidase and an enzyme in fish liver. Though our experiment, following facts were found: the vitamin A oxidizing enzyme in fish liver was a lyo-form and was found not only in mackerel liver but in perch liver. The absorption spectra of vitamin A oxidized by the liver enzyme showed the flattening of peak at 328 mμ in our experiments as same as in the usual by air oxidation. The enzyme possessed a special thermal property as having more activity at 60°C. A large quantity of liver oil inhibited the enzyme activity at room temperature, but at higher temperature the activity began to develop by breaking down of inhibitants. The higher fatty acids seemed to be less oxidized in the states of glyceride by the enzyme. The enzyme had many similar properties to the soybean lipoxidase but showed different value from the soybean enzyme in the optimum pH.