US3104171A - Process for improving the flavor of foods by the addition of 5'-nucleotides - Google Patents

Description

United States Patent 3,104,171 PROCESS FOR lMPRDVlNG THE FLAVGR 6F FOGDS BY THE ADDITIUN OF '-NUCLEOTEDES Kiuichiro Sakaguchi, Tokyo, and Masajiro Kihi and Akira Kunina'ka, Choshishi, Chibaken, Japan, assignors to Yamasa Shoyu KllfrflSliliil Keisha (doing business as Yamasa Sh'oyu Co., Ltd), Choshi, Japan, a Japanese corporation No Drawing. Original application Aug. 22, 1958, Ser. No. 756,541. Divided and this application Dec. 8, 1961, Ser. No. 158,1(98

5 Claims. (ill. 99-140) This invention relates to a process for producing the solution containing 5-nucleotides (adenosine-S'unonophosphate, guanosine 5' Inonophosphate, uridine-S'- monophosphate, cytidine-S'-monophosphate, inosine-5- monophosphate, xanthosine 5 monophosphate) from ribonucleic acid by microbial 5'-phosphodiesterase action, and to an application of the 5-nucleot-ides as special seasonings. The object of this invention is to produce flavorous 5-nucleotides, which were so tar prepared generally only by organic synthesis or by extraction from tissues of various organisms such as mammalian muscle, economically and in good yield from ribonucleic acid, using the enzymes of microorganisms.

This is a division of copending application Serial No. 756,541, filed August 22, 1958.

Chemical degradation of ribonucleic acid results in formation of 3- and 2'-nucleotides and does not result in formation of 5-nucleotides. Furthermore general ribonucleodepolynrerases, without distinction of the kind of origins, degrade ribonucleic acid into 3 (or 2)- nucleotides but not into 5-nucleotides. Only so-calied unspecific phosphodiesterases from snake venom or in testinal mucosa degrade ribonucleic acid into 5'-nucleotides. However, it is very diflicult to obtain a large amount of these enzymes. 5'-nucleotides can be produced by means of organic synthesis but said process is very troublesome and not economical too. Thus, hitherto, the production of 5'-nucleotides was very difficult, and especially economical mass production thereof was quite impossible.

We have found that some strains of bacteria, yeasts, and molds contain 5 '-phosphodiesterases which specifical- 1y hydrolyze the 5'-phosphodiester linkages in ribonucleic acid and produce four 5'-nucleotides: adenosine-S-monophosphate, guanosine 5 monophosphate, cytidine-S-monophosphate, and uridine-5-monophosphate. Especially the several strains which belong to the following genuses have been recognized to contain strong 5'-phosphodiesterase: Bacillus, Streptomyces, Torula, Zygosaccharomyces, Penicillium and Aspergillus. Thus the basis of the production of 5- nucleotides by microorganisms according to the present invention has been established for the first time.

This invention has been accomplished on the basis of the above confirmation. Therefore, the present invention provides a process for the production of 5'-nucleotides which is characterized in this that ribonucleic acid is degraded into Snucleotides by 5-phophodiesterase which is contained in living cells, dry cells, culture filtrates or cell extracts of microorganisms described above. The microorganisms containing 5'-phosphodiesterase are able to be grown on either solid media or liquid media. For economical mass production, however, liquid media are more appropriate. As the components of the culture medium, the conventional carbon and nitrogen sources and several inorganic salts may be employed eifectively.

3,104,171 Patented Sept. 17, 1963 This invention includes both one step method and two step method. In one step method, both growing of microorganism and enzymic degradation of ribonucleic acid are carried out simultaneously, employing culture medium containing ribonucleic acid. In two step method, growing of microorganism and enzyrnic degradation of ribonucleic acid are carried out separately.

According to the present invention, it is not necessary to purify ribonucleic acid before its enzymic degradation. Crude solution containing ribonucleic acid, such as yeast extracts, may be used as an appropriate starting material. Furthermore microbial cells cultivated for producing 5'- phosphodiesterase are eliectively utilized too as a source of ribonucleic acid.

Free 5-nucl'eotides or their alkali salts obtained by the processing as described above enhance or increase the flavor of the foods, beverages, and seasonings in which they are placed. This flavoring action is caused by the synergy between 5-nucleotides and amino acids or organic acid-s. According to our discovery, purine and pyrimidine bases, their nucleosides, and their 2- and 3- nucleotides have little flavor, while 5-nucleotides, especially inosine-5'-monophosphate, guanosine-Y-monophosphate, and xanthosine-S-monophosphate, have very agreeable good taste. Furthermore, there is specific synergy in taste between 5'-nucleotides and amino acids or organic acids. Among various amino acids, glut'arnic and aspartic acids were recognized to be especially elfective in the synergy with 5-nucleotides. General foods, beverages, and seasonings contain considerable quantity of arnino acids or organic acids as main flavoring components, but scarcely contain 5-nucleotides. Therefore, it seems that the role of 5-nucleotides in flavoring is Very important. For example, the good taste of soups or meat extracts, containing small quantity of 5'-nucleotides, may be perhaps caused mainly by the synergy between 5'-nucleotides and amino acids.

This invention relates also to the application of 5- nucleotides based on the utilization of aforesaid synergy between 5'-nucleoti-des and amino acids or organic acids. The application of 5'-nucleotides according to the present invention comprises adding one or more of 5'-nucleotides to general foods or beverages such as meat products, soups, roux, vinegar, various dressings, sauces, curry powder and various drinks including wine, to counteract the displeased pungency which spoils the taste qualities of foods or beverages, and to enhance or increase the flavor specifically according to the synergy between 5'- nucleotides and amino acids or organic acids present in the foods or beverages. In case of application for the foods or beverages containing no amino acids, it is more effective to add monosodium glutamic acid and 5'-nucleotides together. 5-nucleotides may be also employed to enrich specifically the seasonings containing amino acids. The bitter substances in the crude preparations of 5- nucleotides can be readily removed by cation exchange resin. Both crude and purified preparations of 5'-nucleotides are useful.

' According to the present invention, alkali salts of 5'- nucleotides may be also employed similarly as free 5- nucleotides since there is no significant diiference between their flavoring action.

The invention is illustrated but not limited by the following examples.

Example 1 phate, 0.04% of magnesium sulfate, and 0.04% of calcium chloride were sterilized and inoculated with a pure culture of Penicillium citrinum. After surface culture at 3 30 C. for five days the mycelial deck was separated from the culture broth, and washed with sterilized water. The washed mycelial deck was incubated with 50 ml. of 0.5% yeast ribonucleic acid solution containing 0.01 N sodium fluoride at 30 C. After 22.5 hours the deck was removed. The resulting reaction mixture was recognized to contain 70-80 mg. of mononucleotides, 80-90 mg. of nucleosides, and 70-80 mg. of undepolyrnerized polynucleotides. The mononucleotides, which are contained in above mixture, were identified as cytidine-'-monophosphate, adenosine-5'-monophosphate, inosine-5'-monophosphate, uridine-S-rnonophosphate, and guanosine-5'-mono phosphate. The identification was carried out as follows: 23 ml. of .the reaction mixture were adjusted to pH 8.5 with strong sodium hydroxide solution. 2.5 ml. of 20% barium acetate solution were added thereto. The precipitate of barium phosphate formed was removed. The supernatant was adjusted to pH 5.0 with a small quantity of acetic acid. 1 ml. of mercuric acetate solution (20% in 2% acetic acid) was added. The precipitate was centrifuged, washed and suspended in water. Into the suspension hydrogen sulfide gas was introduced to separate nucleotides. The mixture was filtered and the precipitate was washed with hot water. The solution resulting from the washing was combined with the supernatant and a portion of the combined solutions was adjusted to pH 8.5, and charged into anion exchange resin Dowex-l-Cl- X-4 (200-400 mesh) column with a diameter of 1.0 cm. and 23 cm. in height, and was eluted with 0.003 N (No. l-No. 216 test tubes) and 0.010 N hydrogen chloride (No. 217-No. 302 test tubes). Each 80 drops of the eluate were collected into a test tube and optical density at 260 mg of each eluate was read. Five ultraviolet absor'oing fractions A, B, C, D and E were obtained. The

4% growth medium was shaken on a reciprocating shaker at 30 C. After 7 days culture filtrates were concentrated in vacuo, and then dialyzed against running Water overnight. To the dialyzed solution 4 volumes of ethanol were added. The resulting precipitate, which was rich in 5-phosphodiesterase activity, was dried up in a desiccator and was employed as an enzyme preparation. From 1 litre of culture filtrates about 1 g. of the prepanation was obtained. 1 g. of this preparation was incubated with 200 ml. of 5% ribonucleic acid at 65 C. and pH 5.0. Under these conditions phosphomonoesterase and adenyl deanrin'ase were almost inactive, while 5-phosphodiesterase was recognized to be very active.

The reaction proceeds as described below:

Incubation time, min n 0' 10 30' 60} 90 5-nucleutirles formation from ribonucleic acid, percent 68 06 07 Inorg. 1. formation from 5-nuclcoti1les,

percent 0 3. 0 3. 5 4. 5 5. 7

Example 3 100-500 mg. of the crude mixture of adenosine-5'- monophosphate, guanosine-S-monophosphate, cytidinepropenues of these fractions are tabulated as follows: 5'-monophosphate, and urid1ne-5-monophosphate ob- Nueleotidc fraction obtained Standard substance A B C D E Cyti- Adcno- Adeno- Inosine- Urldinc- Guanodinesinesine- 5-mono- 3-monosine- Mixture 013- No of test tubes 3-mono- 3-mono- 5-monophosphos- 3-mouonuclcotidcs phosphosphosphate pliatc phosphate phatc phato pirate 21-24 43-54 121-122 221-237 261-281 Amax (my) 275 258 .249 262 257 278 257 257 253 Percent color, 7 min 78.7 82.2 81. 0 39. G Development, 15 mln. 94.2 98.9 97.2 72.8 (Pentose-), 25 min 100.0 100.0 100.0 04. 7 (Orcinol), 35 min-.. 96.0 06. 8 96.3 99. 0 (Reaction), 45 min. 03. 9 92. 6 92.8 100.0 Carbazolo reaction Blue Blue Blue Purple Blue Purple Distance migrated from origin to anode side by electrophoresis, cm 4.9 4.9 12.5 14.7 7.7 4.9 5.0 4.5 14 8,8. 7,4 7 NaIOm'osaniline reaction.

1 Ultraviolet absorption spectra of standard substances were measured in 0.1 N H01. 1 The technique employed was essentially the same as that described by Albaum and Umbreit (J. Biol. Chem, 167, 360, (1047)). 8 Starting line was at 5 cm. from the end on the cathode side, and 26 cm. from the end on the anode side.

For the formation of the strong 5-phosphodiesterase, shaking culture is more eiiective than surface culture at least in case of the strain employed in Example 1.

The culture medium employed in Exarnple 1 was inoculated with Penicillium citrinum. The inoculated tained in Example 2 were added to 1 litre of sauce. (Practically, 2-10 ml. of reaction mixture were employed against 1 litre of sauce.) By this treatment displeased pungency of sauce was counteracted perfectly, and the good flavor was greatly increased. Thus the sauce became fiavorous almost beyond recognition. The mixture of deaminated 5-nucleotides containing inosine-5'-monophosphate, xanthosine-5-monophosphate, and uridine-5- monophosphate, was also employed to improve the taste of sauce. The mixture of 5'-nucleotides was able to be kept in a form of dry powder for a long time.

Example 4 Monosodium glutamate was coated with purified inosine-5'monophosphate disodium salt or guanosine-5'- monophosphoric acid. The ratio of monosodium glutamate to inosine-S'-monophosphate or guanosine-5'-monophosphate was 5-15:1. The resultant superior seasoning was recognized to have remarkable flavoring properties for all kind of dishes.

Example T o 120 g. of soup potage powder (corresponding to 1800 ml. of fin'al volume) 1 to 2 g. of purified inosine-5'- monophosphaite disodium salt or guanosine-5-monophosphoric acid were added. From the resultant enriched powder, remarkable fiavorous soup was prepared. Instead of the purified nucleotide preparations each crude preparation or mixture of 5-n-uoleotides was also employed satisfactorily.

In case of soup consom-me being prepared inos=ine5- monophosphate, guanosine-S-rnonophosphate, and mixture of 5-nucleotides were employed effectively too.

What is claimed is:

1. A process for improving and increasing specifically the flavor of foods, beverages, and seasonings containing amino acids which comprises adding thereto at least one compound selected from the group consisting of free 5'- nucleotides and their alkali salts.

2. A process for improving and increasing specifically the flavor of foods, beverages, and seasonings containing substantially no amino acids which comprises adding thereto at least one 5'-nucleotide in addition to mono sodium glutamate.

3. A process for preparing 5 -nucleotides which comprises cultivating the microorganism selected from the group consisting of Bacillus, Streptomyces, Torula, Zygosaccharomyces, Aspergillus and Penicillium which contain 5-phosphodiesterase, degrading ribonucleic acid to 5'-nucleotides by 5'phosphodiestenase which is contained in the cultured material selected from the group consisting of living cells, dry cells, culture filtrates and cell extracts resulting fronr the cultivation of said microorganism, and adding said 5'-nucleotides to a material selected from the class consisting of foods, beverages and seasonings containing amino acids.

4. A process for preparing 5-nucleotides which comprises cultivating the microorganism selected from the group consisting of Bacillus, Streptomyces, Torula, Zygosaccharomyces, Aspergillus and Penicillium which contain 5-phosphodiesterase, degrading ribonucleic acid to 5'-nucleotides by 5'-phosphodiesterase which is contained in the cultured material selected from the group consisting of living cells, dry cells, culture filtrates and cell extracts resulting from the cultivation of said microorganism, adding an alkali salt to a solution containing said 5'-nucleotides and admixing the solution with a material selected from the class consisting of foods, beverages and seasonings containing amino acids.

5. A process for preparing 5-nucleotides Which comprises cultivating the microorganism selected from the group consisting of Bacillus, Streptomyces, T orula, Zygosaccharomyces, Aspergillus and Penicillium which contain 5'-phosphodiesterase, degrading ribonucleic acid to References Cited in the file of this patent Cohn et al.: Journal of Biological Chemistry, vol. 203, July-August 1953, pages 319 to 331.

Dixon et al.: Enzymes, published by Academic Press Inc., New York, 1958, pages 190, 191 and 690.

Claims (1)

1. A PROCESS FOR IMPROVING AND INCREASING SPECIFICALLY THE FLAVOR OF FOODS, BEVERAGES, AND SEASONINGS CONTAINING AMINO ACIDS WHICH COMPRISES ADDING THERETO AT LEAST ONE COMPOUND SELECTED FROM THE GROUP CONSISTING OF FREE 5''NUCLEOTIDES AND THEIR ALKALI SALTS.