Stability of RIBOTIDE

Effectt of pH and heattiing Effec of pH and hea ng

The experiments were carried out under conditions of acid, neutral and alkaline pH and heating at 100C and 120C, respectively. A 0.02% aqueous solution of RIBOTIDE and a 1mM (about 0.05%) solution respectively of 5-inosinic acid and 5-guanylic acid were prepared and were adjusted to pH 3, 4, 5, 7 and 9. The test solutions were placed in ampoules, and heated at 100C over a boiling water bath or at 120C in an autoclave under increased pressure. In Figs. 1-1 to 3-2 are shown the residual levels of RIBOTIDE, 5-inosinic acid and 5-guanylic acid. In the experiments, it was found that RIBOTIDE, 5-inosinic acid and 5-guanylic acid respectively remained at a relatively high level in the solutions adjusted to pH 3 and heated at 100C but were decomposed to a considerable extent when heated at 120C, and that the decomposition was more prominent when heated for a longer time, apparently indicating that higher temperature and longer heating accelerated the breakdown of 5-ribonucleotide. It was observed, however, that the substances were considerably more stable in the solutions adjusted to pH 5 or higher, and were less affected by heating as the pH rose from neutral to alkaline, e.g., 82% of RIBOTIDE was found in the solution adjusted to pH 7 which had been subjected to such severe conditions as heating at 120C for 60 minutes. It was also found that, of the three test substances, 5-inosinic acid was slightly more thermolabile (readily changed or destroyed by heat) as a whole.
Fig. 1-1: Effect of pH and heating on Ribotide
100% Remaining level 80% 60% 40% 20% 0% 0 20 40 60 pH3 (acetic acid) heating time at 100 C (in minutes) pH7 pH5 pH3 (HCl) pH9

Fig 1-2: Effect of pH and heating on Ribotide

100% Remaining level 80% 60% 40% 20% 0% 0 20 40 60 pH3 (acetic acid) heating time at 120 C (in minutes) pH7 pH5 pH3 (HCl) pH9

Fig. 2-1: Effect of pH and heating on Inosinic acid

100% Remaining level 80% 60% 40% 20% 0% 0 20 40 60 heating time at 100 C (in minutes) Remaining level pH3 (HCl) pH3 (acetic acid)

Fig. 2-2: Effect of pH and heating on Inosinic acid

100% 80% 60% 40% 20% 0% 0 20 40 60 heating time at 120 C (in minutes) pH3 (acetic acid) pH5 pH3 (HCl) pH7

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Fig. 3-1: Effect of pH and heating on Guanylic acid

100% Remaining level 80% 60% 40% 20% 0% 0 20 40 60 heating time at 100 C (in minutes)

Fig. 3-2: Effect of pH and heating on Guanylic acid

100% Remaining level

pH3 (HCl) pH3 (acetic acid)

80% 60% 40% 20% 0% 0 20 40 60 heating time at 120 C (in minutes)

pH7 pH5 pH3 (HCl) pH3 (acetic acid)

Effectt of substtances commonlly presentt Effec of subs ances common y presen

In an attempt to prove the effect of substances which are commonly present, RIBOTIDE was added at a 0.02% level to solutions of seasoning agents and other food additives and to other solutions of various substances as shown in the table below as well as addition in a 0.02% concentration to solutions of acids such as hydrochloric acid, acetic acid, succinic acid and citric acid, and these solutions were adjusted to pH 3 and heated at 100C and 120C under increased pressure. The same heating and pressurising method was employed as for pH and heating test above.
Salt MSG Sucrose Sucrose Glucose HCl Substances commonly present Concentration 3% 0.3% 3% 50% 3% 1/1000N pH of solution 3 100C 20 min. 98 100 97 100 (30) 99 97 100C 40 min. 98 100 96 100 93 96 97 100C 60 min. 97 100 96 96 89 91 91 120C 20 min. 90 91 79 83 69 120C 40 min. 87 84 69 78 60 120C 60 min. 85 82 68 72 58 RIBOTIDE addition level: 0.02% (The remaining level indicated by %) Acetic acid Succinic Citric acid acid Fe+++

1% 3 99 89 86 -

1/100M 1/500M 3 3 100 92 98 89 92 85 -

10ppm 85 76

There was no appreciable difference in the residual RIBOTIDE level between the RIBOTIDE solution free from other substances and that containing the substances when heated at 100C, but a slight difference was noted between the solutions when heated at 120C according to the type and concentration of such substances commonly present, though it was observed that no pronounced breakdown of RIBOTIDE was caused in a 3% sodium chloride solution, a FeC14 solution containing 10 p.p.m. of Fe ion and a 3% sucrose solution. The breakdown of RIBOTIDE, however, appeared to be slightly accelerated in a 3% glucose solution, 50% sucrose solution and 0.3% sodium glutamate solution and it appeared to be quite rapidly decomposed in a 3% glucose solution when heated at 120C. On the other hand, no large difference was noted in the stability of RIBOTIDE to various acids such as hydrochloric acid, acetic acid, succinic acid and citric acid in the respective solutions adjusted to the same pH value when heated at 100C. Since these acids are added to foods at a considerably high level, however, it will be necessary to examine the effect of these acids on RIBOTIDE under more practical conditions.

Heating condition

Ribotide Stability

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Summary Summary
Many of the daily foods are neutral to slightly acid to litmus, being in a pH range of 4-6, and are never in a strong acid range of below pH 3 nor alkaline above pH 9. It also appears that the heating temperature usually employed for food processing, such as thermal treatment, boiling and steaming (*1), is in a range of 100C to 110C and, only few of the processed foods are heated for longer than 60 minutes. Even in the cooking of a few variety of food where other substances are commonly present at a high level and which are heated for a relatively long time, e.g., preserved foods boiled in soy sauce, other materials are first boiled sufficiently in a seasoning liquid and are finished with another seasoning liquid containing RIBOTIDE, i.e., so-called sprinkling liquid, so that RIBOTIDE so added is scarcely heated under increased pressure or heated only for an extremely short time. From the results of the present experiments, it appears hardly possible that RIBOTIDE is decomposed and loses its taste-improving property even when it is added to food before boiling or even by sterilization at 115C for 15 minutes. Since even processed foods, such as soup stock, curry bar, instantly ready Chinese noodles, soy sauce, etc. where the addition of RIBOTIDE brings out a prominent taste-improving effect, are subjected to heating at 100C or higher only for an extremely short time, it may be considered that RIBOTIDE is not seriously affected by salt, sugars and acids which are commonly present during the processing. Breakdown of RIBOTIDE, takes place rapidly in fermentation food products or fresh foods where phosphatase is present and to which RIBOTIDE is added without or before heating. It has been found in other cases, however that RIBOTIDE is only slightly decomposed during the processing of food.
(Note *1) Data for frying in oil is not available. Temperature of foodstuff in frying is not so high as oil temperature and heating time is short. Frying for a long time will result in black burning of foodstuff. Ribotide is commonly used in fried foods such as Chinese stir-fry and Japanese tempura for which foodstuff is fried at more than 180C.

Effectt of Miicrowave Cookiing Effec of M crowave Cook ng

1. Specimen : Solutions
(1) (2) Solution 1 Solution 2 : : Aqueous solution of about 0.1% RIBOTIDE Aqueous solution of about 0.1% RIBOTIDE containing 1% of salt and 1% of sucrose (pH5.5)

2.

Microwave cooking
(1) (2) (3) (4) Pour 20ml of each of solutions in to 50ml volumetric flask. Heat the solution in a microwave oven (Hitachi MR-T500, 950W) for 2, 5 and 10 min. Fill up the volume of the solution to 50ml with water. Determine the contents of nucleotides by HPLC.

3.

Results (Remaining rate (%) of nucleotides)

Solution 1 Solution 2 Inosinate Guanylate Inosinate Guanylate 2 min. 100% 99% 99% 99% Heating time 5 min. 98% 98% 99% 97% 10 min. 98% 98% 97% 96%

4.

Conclusion
RIBOTIDE in aqueous solution is stable against microwave cooking.

Ribotide Stability

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