METHOD OF PRODUCING FLAVOR BY MIXED FERMENTATION OF HETEROLOGOUS MICROORGANISMS

Abstract

The present invention relates to a method of producing a flavor by a co-fermentation process using mixed fermentation of two or more different microorganisms producing different products. The method of producing a flavor may produce a natural flavor capable of improving the taste and aroma of food and the overall sensory properties of food through a fermentation broth containing amino acids, nucleic acids and/or organic acids, which is produced by mixed fermentation of different microorganisms producing different products, that is, different kinds of amino acids, nucleic acids and/or organic acids. This flavor may be used in various food fields.

Claims

1. A method for producing a flavor, the method comprising a step of inoculating a fermentation medium with a first microorganism and a second microorganism and then producing a fermentation broth containing amino acid, nucleic acid and/or organic acid by fermentation of the microorganisms, wherein the first microorganism and the second microorganism produce different products and each produces one selected from the group consisting of amino acid, nucleic acid and organic acid.

2. The method of claim 1, wherein the amino acid is at least one selected from the group consisting of L-glutamic acid, L-alanine, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, L-glycine, L-serine, L-threonine, L-cysteine, L-asparagine, L-glutamine, L-aspartic acid, L-lysine, L-arginine, and L-histidine.

3. The method of claim 1, wherein the nucleic acid is at least one selected from the group consisting of inosinic acid, guanylic acid, xanthylic acid, and salts thereof.

4. The method of claim 1, wherein the organic acid is at least one selected from the group consisting of succinic acid, malic acid, citric acid, acetic acid, lactic acid, fumaric acid, tartaric acid, ascorbic acid, gluconic acid, and salts thereof.

5. The method of claim 1, wherein the first microorganism is a glutamic acid-producing microorganism, and the second microorganism is a lysine-producing microorganism, an arginine-producing microorganism, a histidine-producing microorganism, a tryptophan-producing microorganism, a glycine-producing microorganism, an alanine-producing microorganism, a succinic acid-producing microorganism, a lactic acid-producing microorganism, a guanylic acid-producing microorganism, or an inosinic acid-producing microorganism.

6. The method of claim 1, wherein the first microorganism is an inosinic acid-producing microorganism, and the second microorganism is a lysine-producing microorganism, an arginine-producing microorganism, a histidine-producing microorganism, a tryptophan-producing microorganism, a glycine-producing microorganism, an alanine-producing microorganism, a succinic acid-producing microorganism, a lactic acid-producing microorganism, or a guanylic acid-producing microorganism.

7. The method of claim 1, wherein the step comprises further inoculating the fermentation medium with a third microorganism, which produces a product different from products produced from the first microorganism and the second microorganism and produces one selected from the group consisting of amino acid, nucleic acid and organic acid.

8. The method of claim 7, wherein the first microorganism is a glutamic acid-producing microorganism, the second microorganism is a guanylic acid-producing microorganism, the third microorganism is an inosinic acid-producing microorganism.

9. The method of claim 7, wherein the first microorganism, the second microorganism and the third microorganism are microorganisms of the genus Corynebacterium.

10. The method of claim 7, wherein the first microorganism, the second microorganism and the third microorganism are in a seed culture broth state obtained by individual culture or co-culture.

11. The method of claim 7, wherein the step comprises adjusting the inoculum of each microorganism in order to control the ratio between the products of the microorganisms in the fermentation broth.

12. The method of claim 7, wherein the fermentation broth contains the products of the microorganisms in an amount of 3 to 90 wt % relative to the total solid content of the fermentation broth.

13. A method for producing a flavor containing L-glutamic acid and L-lysine, the method comprising a step of inoculating a fermentation medium with a glutamic acid-producing microorganism and a lysine-producing microorganism and then producing a fermentation broth containing L-glutamic acid and L-lysine by fermentation of the microorganisms.

14. The method of claim 13, wherein the glutamic acid-producing microorganism and the lysine-producing microorganism are microorganisms of the genus Corynebacterium.

15. The method of claim 13, wherein the glutamic acid-producing microorganism and the lysine-producing microorganism are in a seed culture broth state obtained by individual culture or co-culture.

16. The method of claim 13, wherein the step comprises adjusting the inoculum of each of the glutamic acid-producing microorganism and the lysine-producing microorganism in order to control the ratio of L-glutamic acid to L-lysine in the fermentation broth.

17. The method of claim 13, wherein the fermentation broth contains amino acids, including L-glutamic acid and L-lysine, in an amount of 3 to 90 wt % relative to the total solid content of the fermentation broth.

18. A flavor produced by the method of claim 7.

19. A flavor containing L-glutamic acid and L-lysine, produced by the method of claim 13.

20. The flavor of claim 19, containing L-glutamic acid and L-lysine in an amount of 3 to 90 wt % relative to the total solid content of the flavor.

21. A food composition containing the flavor of claim 18.

22. A food composition containing the flavor containing L-glutamic acid and L-lysine according to claim 19.

23. The method of claim 1, wherein the first microorganism and the second microorganism are microorganisms of the genus Corynebacterium.

24. The method of claim 1, wherein the first microorganism and the second microorganism are in a seed culture broth state obtained by individual culture or co-culture.

25. The method of claim 1, wherein the step comprises adjusting the inoculum of each microorganism in order to control the ratio between the products of the microorganisms in the fermentation broth.

26. The method of claim 1, wherein the fermentation broth contains the products of the microorganisms in an amount of 3 to 90 wt % relative to the total solid content of the fermentation broth.

27. A flavor produced by the method of claim 1.

28. A food composition containing the flavor of claim 27.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0121] FIG. 1 is a flow chart showing a fermentation process performed using a glutamic acid-producing microorganism and a lysine-producing microorganism in Preparation Examples 1 to 3 according to one embodiment of the present invention.

[0122] FIG. 2 is a flow chart showing a fermentation process performed using a glutamic acid-producing microorganism and an arginine-producing microorganism in Preparation Examples 4 to 6 according to one embodiment of the present invention.

[0123] FIG. 3 is a flow chart showing a fermentation process performed using a glutamic acid-producing microorganism and an inosinic acid-producing microorganism in Preparation Examples 7 to 9 according to one embodiment of the present invention.

[0124] FIG. 4 is a flow chart showing a fermentation process performed using an inosinic acid-producing microorganism and a lysine-producing microorganism in Preparation Examples 10 to 12 according to one embodiment of the present invention.

[0125] FIG. 5 is a flow chart showing a fermentation process performed using an inosinic acid-producing microorganism and an arginine-producing microorganism in Preparation Examples 13 to 15 according to one embodiment of the present invention.

DETAILED DESCRIPTION

[0126] Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

Example 1. Mixed Fermentation of Glutamic Acid and Lysine

[0127] 1-1. Seed Culture

[0128] Corynebacterium glutamicum NFG6 (KCCM13164P) that produces L-glutamic acid (GA) was used as a glutamic acid-producing microorganism, and Corynebacterium glutamicum NFL21 (KCCM13163P) that produces L-lysine (LYS) was used as a lysine-producing microorganism.

[0129] For seed culture of the glutamic acid-producing microorganism, the glutamic acid-producing microorganism was inoculated into a 2-L flask containing 0.2 L of a seed culture medium, followed by primary culture for 22 to 24 hours at 30° C. and 140 rpm (OD.sub.610=10 to 15). Next, 2 to 3% of the primary culture was inoculated into a 5-L jar fermenter, and 2 to 2.5 L of a seed culture medium was added thereto, followed by secondary culture at 32° C., pH 6.9, 600 rpm and an aeration rate of 1.0 vvm for 22 to 24 hours (OD.sub.610=20 to 60), thereby preparing a glutamic acid seed culture broth.

[0130] For seed culture of the lysine-producing microorganism, the lysine-producing microorganism was inoculated into a 2-L flask containing 0.2 L of a seed culture medium, followed by primary culture for 16 to 18 hours at 30° C. and 140 rpm (OD.sub.610=11 to 12). Next, 5% of the primary culture was inoculated into a 5-L jar fermenter, and 2 to 2.5 L of a seed culture medium was added thereto, followed by secondary culture at 32° C., pH 7.0, 650 rpm and an aeration rate of 2.0 vvm for 21 to 24 hours (OD.sub.610=20 to 60), thereby preparing a lysine seed culture broth.

[0131] The compositions of the seed culture media used are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Composition Seed culture 4.5 to 5.5% molasses, 3% glucose, 0.85% yeast medium for extract paste, 100 ppm methionine, 0.6% H.sub.3PO.sub.4, glutamic acid- 0.1% sodium succinate, 50 ppm vitamin C, 12 producing ppm thiamine HCl, 20 ppb vitamin B12, 10 ppm microorganism biotin, 0.4% MgSO.sub.4, and 0.01% antifoaming agent for food Seed culture 1.5 to 3% molasses, 9 to 12% raw sugar, 1% medium for yeast extract paste, 1.6% (NH.sub.4).sub.2SO.sub.4, 0.3% H.sub.3PO.sub.4, lysine-producing 7.3 ppm MnSO.sub.4•5H.sub.2O, 14 ppm nicotinamide, 2.5 microorganism ppm thiamine HCl, 1.5 ppm CuSO.sub.4•5H.sub.2O, 0.056 ppm biotin, 0.045% betaine, and 0.01% antifoaming agent for food

[0132] 1-2. Main Fermentation

[0133] In order to examine the ratio of glutamic acid to lysine in the fermentation broth depending on the inoculums of the glutamic acid seed culture broth and the lysine seed culture broth, the glutamic acid seed culture broth and the lysine seed culture broth were inoculated at various ratios and fermented.

[0134] In main fermentation, 14 to 18 L of a fermentation medium was added to a 50 L fermenter, and inoculated with 1.2 to 1.8 L of the total seed culture broth at an inoculation ratio of 50 to 99.95 (glutamic acid seed culture broth): 50 to 0.05 (lysine seed culture broth), followed by mixed fermentation by fed-batch culture for 28 to 40 hours. The fermentation medium composition and fermentation conditions used are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Fermentation medium 1.5 to 3% molasses, 2.5 to 4% glucose, 0.4 to composition 1% yeast extract paste, 0.1 to 0.2% H.sub.3PO.sub.4, 0.05 to 0.12% betaine, and 0.005% antifoaming agent for food Fermentation Temperature 32.fwdarw.38° C., pH 6.5 to 7.5, aeration conditions rate 0.8 to 1.2 vvm, internal pressure 0.6 to 1.0 kg/cm.sup.3, agitation speed 320 to 350 rpm, and dissolved oxygen (DO) concentration 20 to 70%

[0135] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Inoculation ratio GA:LYS ratio between seed culture Fermentation in fermentation broths (GA:LYS) time broth 99.95:0.05  31 hours .sup. 99:1 75:25 29 hours 1.48:1 70:30 29 hours 1.30:1 67:33 30 hours 1.10:1 65:35 30 hours 1.01:1 60:40 29 hours 0.93:1 50:50 28 hours 0.83:1

[0136] Referring to Table 3 above, it was confirmed that, when the seed culture broths of the amino acid-producing microorganisms were inoculated at a ratio of 50 to 99.95 (glutamic acid-producing microorganism):50 to 0.05 (lysine-producing microorganism), L-glutamic acid and L-lysine in the fermentation broth were produced at a ratio of 0.83 to 99:1.

Example 2. Comparison of Glutamic Acid-Lysine Fermented Powders Between Fermentation Processes

[0137] 2-1. Production of Glutamic Acid-Lysine Fermented Powder

[0138] Conventionally, a flavor containing glutamic acid and lysine was produced by individually fermenting a glutamic acid-producing microorganism and a lysine-producing microorganism and then mixing the amino acid-containing fermentation broths or dried products thereof at a suitable ratio. To compare the differences in taste between this conventional individual fermentation method and the method based on mixed fermentation of the glutamic acid-producing microorganism and the lysine-producing microorganism, the components of glutamic acid-lysine (GA-LYS) fermented powders obtained by the production methods were compared. (See FIG. 1).

[0139] The glutamic acid seed culture broth and lysine seed culture broth used here were prepared in the same manner as in Example 1-1.

{circle around (1)} Individual Fermentation (Preparation Example 1)

[0140] In the method of mixing individual dried products among the conventional methods, each of the glutamic acid seed culture broth and the lysine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. Next, cells were separated from each fermentation broth, and then decolorization and filtration processes were performed. The filtrates were concentrated and dried to obtain dried products. The obtained dried glutamic acid and lysine products were mixed together so that the ratio of glutamic acid to lysine was 1:1, thereby preparing fermented powder containing glutamic acid and lysine.

{circle around (2)} Mixing of Fermentation Broths after Individual Fermentation (Preparation Example 2)

[0141] In the method of mixing individual fermentation broths among the conventional methods, each of the glutamic acid seed culture broth and the lysine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. The fermentation broths obtained in the main fermentation were mixed together so that the ratio of glutamic acid to lysine was 1:1. Next, cells were separated from the fermentation broth mixture, and then decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-lysine fermented powder.

{circle around (3)} Mixed Fermentation (Preparation Example 3)

[0142] In mixed fermentation, the glutamic acid seed culture broth and the lysine seed culture broth were inoculated at a ratio of 65:35 in the same manner as in Example 1-2 so that the ratio of glutamic acid to lysine in the fermentation broth was about 1:1 in the same manner as in Preparation Examples 1 and 2, followed by mixed fermentation. Next, cells were separated from the fermentation broth, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-lysine fermented powder.

[0143] 2-2. Comparison of Components Between Glutamic Acid-Lysine Fermented Powders

[0144] Component analysis was performed on the glutamic acid-lysine fermented powder obtained through individual fermentation in each of Preparation Examples 1 and 2 and the glutamic acid-lysine fermented powder obtained through mixed fermentation in Preparation Example 3.

[0145] Amino acids (L-glutamic acid and L-lysine) were measured by HPLC analysis (GA—210 nm, UV detector, flow rate 0.9 ml/min; LYS—214 nm, UV detector, flow rate 0.8 ml/min), and organic acids (citric acid, succinic acid, lactic acid, acetic acid, etc.) were measured by HPLC analysis (HPX-87H column, 214 nm, 25 min). Ions (Na, Mg, K, PO.sub.4, SO.sub.4, Cl, NH.sub.4, etc.) were measured by an ion analyzer (Dionex IcS-1100, Thermo Scientific). The results are shown in Table 4 below.

TABLE-US-00004 TABLE 4 Preparation Preparation Preparation Item Example 1 Example 2 Example 3 GA  22% 35% 43% LYS  22% 35% 43% AA/TS*  47% 72% 88% Total nitrogen 9.0% 12.2%.sup.  11.8%.sup.  Organic acids 3.1% 2.6%  1.5%  Ions 8.1% 10.2%.sup.   2% Ammonium 3.8% 2.5%  0.7%  *AA/TS: proportion of amino acids (GA + LYS) relative to total solid content

[0146] Referring to Table 4 above, mixed fermentation (Preparation Example 3) showed a higher amino acid proportion than individual fermentation (Preparation Examples 1 and 2) and showed significant decreases in the contents of organic acids, ions and ammonium.

[0147] 2-3. Sensory Comparison Between Individual Fermentation and Mixed Fermentation

[0148] Sensory evaluation was performed on the glutamic acid-lysine fermented powders obtained through individual fermentation in Preparation Examples 1 and 2 and the glutamic acid-lysine fermented powder obtained through mixed fermentation in Preparation Example 3.

[0149] For sensory evaluation of the glutamic acid-lysine fermented powders, the umami taste, umami persistence, salty taste, sour taste, bitter taste and sweet taste of each powder itself or a sample obtained by diluting each powder in lukewarm water at a concentration of 1 to 5% were evaluated by a trained panel consisting of 10 to 15 persons. The results are shown in Table 5 below.

TABLE-US-00005 TABLE 5 Preparation Preparation Preparation Example 1 Example 2 Example 3 (individual (individual (mixed Item fermentation) fermentation) fermentation) Initial Umami taste +++ ++++ ++++ Umami persistence ++ +++ ++++ Salty taste − + ++ Sour taste ++ + − Bitter taste ++ + − Sweet taste + + ++

[0150] Referring to Table 5 above, due to the difference in components of the glutamic acid-lysine fermented powder as shown in Table 4 above, the sample prepared by the mixing process after individual fermentation showed a weak umami taste and an increased bitter taste and sour taste compared to the sample prepared by mixed fermentation. In addition, since the increase in by-products such as organic acids and the increase in ions as shown in Table 4 above also affect the sensory properties of the sample, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after individual fermentation.

Example 3. Mixed Fermentation of Glutamic Acid and Arginine

[0151] 3-1. Seed Culture

[0152] Corynebacterium glutamicum NFG6 (KCCM13164P) was used as a glutamic acid-producing microorganism, and Corynebacterium glutamicum NFA40 (KCCM13165P) that produces L-arginine (ARG) was used as an arginine-producing microorganism.

[0153] A glutamic acid seed culture broth of the glutamic acid-producing microorganism was prepared in the same manner as in Example 1-1.

[0154] For seed culture of the arginine-producing microorganism, the arginine-producing microorganism was inoculated into a 2-L flask containing 0.2 L of a seed culture medium, followed by primary culture for 16 to 18 hours at 30° C. and 140 rpm (OD.sub.610=12 to 18). Next, 2 to 4% of the primary culture was inoculated into a 5-L jar fermenter, and 2 to 2.5 L of a seed culture medium was added thereto, followed by secondary culture at 32° C., pH 6.7, 600 rpm and an aeration rate of 1.0 vvm for 16 to 24 hours (OD.sub.610=20 to 60), thereby preparing an arginine seed culture broth. The composition of the seed culture medium used is shown in Table 6 below.

TABLE-US-00006 TABLE 6 Composition Seed culture 1.5 to 4.5% glucose, 2 to 6% raw sugar, 2 to medium for 3% yeast extract paste, 0.6% (NH.sub.4).sub.2SO.sub.4, 0.2% arginine- KH.sub.2PO.sub.4, 0.2% K.sub.2HPO.sub.4 0.2%, 15 ppm MnSO.sub.4•5H.sub.2O, producing 0.2% MgSO4•7H.sub.2O 1 ppm thiamine HCl, 10 ppm microorganism ZnSO.sub.4•7H.sub.2O, 0.3 ppm biotin, 15 ppm FeSO.sub.4•7H.sub.2O, and 0.01% antifoaming agent for food

[0155] 3-2. Main Fermentation

[0156] In order to examine the ratio of glutamic acid to arginine in the fermentation broth depending on the inoculums of the glutamic acid seed culture broth and the arginine seed culture broth, the glutamic acid seed culture broth and the arginine seed culture broth were inoculated at various ratios and fermented.

[0157] In main fermentation, 14 to 18 L of a fermentation medium was added to a 50-L fermenter and inoculated with 1.2 to 1.8 L of the total seed culture broth at an inoculation ratio of 30 to 99.95 (glutamic acid seed culture broth): 70 to 0.05 (arginine seed culture broth), followed by mixed fermentation by fed-batch culture for 31 to 60 hours. The fermentation medium composition and fermentation conditions used are shown in Table 7 below.

TABLE-US-00007 TABLE 7 Fermentation 1.5 to 3% molasses, 2.5 to 4% glucose, 0.4 to 1% medium yeast extract paste, 0.05 to 0.3% (NH.sub.4).sub.2SO.sub.4, 0.1 to composition 0.2% H.sub.3PO.sub.4, 0.05 to 0.12% betaine, and 0.005% antifoaming agent for food Fermentation Temperature 32 to 37° C., pH 6.5 to 7.5, aeration conditions rate 0.8 to 1.2 vvm, internal pressure 0.5 to 1.0 kg/cm.sup.3, agitation speed 320 to 350 rpm, and dissolved oxygen (DO) concentration 20 to 70%

[0158] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 8 below.

TABLE-US-00008 TABLE 8 Inoculation ratio GA:ARG ratio between seed culture Fermentation in fermentation broths (GA:ARG) time broth 99.95:0.05  31 hours 99.0:1 90:10 31 hours 12.2:1 80:20 34 hours 4.36:1 70:30 36 hours 2.41:1 60:40 38 hours 1.96:1 50:50 40 hours 1.57:1 40:60 44 hours 1.21:1 30:70 45 hours 1.04:1

[0159] Referring to Table 8 above, it was confirmed that, when the seed culture broths of the amino acid-producing microorganisms were inoculated at a ratio of 30 to 99.95 (glutamic acid-producing microorganism):70 to 0.05 (arginine-producing microorganism), L-glutamic acid and L-arginine in the fermentation broth were produced at a ratio of 1.04 to 99:1.

Example 4. Comparison of Glutamic Acid-Arginine Fermented Powders Between Fermentation Processes

[0160] 4-1. Production of Glutamic Acid-Arginine Fermented Powder

[0161] Conventionally, a flavor containing glutamic acid and arginine was produced by individually fermenting a glutamic acid-producing microorganism and an arginine-producing microorganism and then mixing the amino acid-containing fermentation broths or dried products thereof at a suitable ratio. To compare the differences in taste between this conventional individual fermentation method and the method based on mixed fermentation of the glutamic acid-producing microorganism and the arginine-producing microorganism, the components of glutamic acid-arginine (GA-ARG) fermented powders obtained by the production methods were compared (see FIG. 2).

[0162] The glutamic acid seed culture broth and arginine seed culture broth used here were prepared in the same manner as in Example 3-1.

{circle around (1)} Individual Fermentation (Preparation Example 4)

[0163] In the method of mixing individual dried products among the conventional methods, each of the glutamic acid seed culture broth and the arginine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. Next, cells were separated from each fermentation broth, and then decolorization and filtration processes were performed. The filtrates were concentrated and dried to obtain dried products. The obtained dried glutamic acid and arginine products were mixed together so that the ratio of glutamic acid to arginine was 1:1, thereby preparing fermented powder containing glutamic acid and arginine.

{circle around (2)} Mixing of Fermentation Broths after Individual Fermentation (Preparation Example 5)

[0164] In the method of mixing individual fermentation broths among the conventional methods, each of the glutamic acid seed culture broth and the arginine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. The fermentation broths obtained in the main fermentation were mixed together so that the ratio of glutamic acid to arginine was 1:1. Next, cells were separated from the fermentation broth mixture, and then decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-arginine fermented powder.

{circle around (3)} Mixed Fermentation (Preparation Example 6)

[0165] In mixed fermentation, the glutamic acid seed culture broth and the arginine seed culture broth were inoculated at a ratio of 30:70 in the same manner as in Example 3-2 so that the ratio of glutamic acid to arginine in the fermentation broth was about 1:1 in the same manner as in Preparation Examples 4 and 5, followed by mixed fermentation. Next, cells were separated from the fermentation broth, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-arginine fermented powder.

[0166] 4-2. Comparison of Components Between Glutamic Acid-Arginine Fermented Powders

[0167] Component analysis was performed on the glutamic acid-arginine fermented powder obtained through individual fermentation in each of Preparation Examples 4 and 5 and the glutamic acid-arginine fermented powder obtained through mixed fermentation in Preparation Example 6.

[0168] Amino acids (L-glutamic acid and L-arginine) were measured by HPLC analysis (GA—210 nm, UV detector, flow rate 0.9 ml/min; ARG—195 nm, UV detector, flow rate 1 ml/min). Organic acid and ions were measured in the same manner as in Example 2-2. The results are shown in Table 9 below.

TABLE-US-00009 TABLE 9 Preparation Preparation Preparation Item Example 4 Example 5 Example 6 GA  22%  35%  42% ARG  22%  34%  42% AA/TS*  48%  71%  86% Total nitrogen 9.4% 12.0%  11.7%  Organic acids 3.6% 2.5% 1.8% Ions 8.3% 9.9% 2.1% Ammonium 4.0% 2.6% 0.9% *AA/TS: proportion of amino acids (GA + ARG) relative to total solid content

[0169] Referring to Table 9 above, mixed fermentation (Preparation Example 6) showed a higher amino acid proportion than individual fermentation (Preparation Examples 4 and 5) and showed significant decreases in the contents of organic acids, ions and ammonium.

[0170] 4-3. Sensory Comparison Between Individual Fermentation and Mixed Fermentation

[0171] Sensory evaluation was performed on the glutamic acid-arginine fermented powders obtained through individual fermentation in Preparation Examples 4 and 5 and the glutamic acid-arginine fermented powder obtained through mixed fermentation in Preparation Example 6.

[0172] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 10 below.

TABLE-US-00010 TABLE 10 Preparation Preparation Preparation Example 4 Example 5 Example 6 (individual (individual (mixed Item fermentation) fermentation) fermentation) Initial Umami taste ++ +++ ++++ Umami persistence + ++ ++++ Salty taste − + ++ Sour taste + + − Bitter taste ++ ++ − Sweet taste − − −

[0173] Referring to Table 10 above, due to the difference in components of the glutamic acid-arginine fermented powder as shown in Table 9 above, the sample prepared by the mixing process after individual fermentation showed a weak umami taste and an increased bitter taste compared to the sample prepared by mixed fermentation. In addition, since the increase in by-products such as organic acids and the increase in ions as shown in Table 9 above also affect the sensory properties of the sample, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after individual fermentation.

Example 5. Mixed Fermentation of Glutamic Acid and Inosinic Acid

[0174] 5-1. Seed Culture

[0175] Corynebacterium glutamicum NFG6 (KCCM13164P) was used as a glutamic acid-producing microorganism, and Corynebacterium ammoniagenes NFI545 (KCCM13162P) that produces IMP was used as an inosinic acid-producing microorganism.

[0176] A glutamic acid seed culture broth of the glutamic acid-producing microorganism was prepared in the same manner as in Example 1-1.

[0177] For seed culture of the inosinic acid-producing microorganism, the inosinic acid-producing microorganism was inoculated into a 2-L flask containing 0.3 L of a seed culture medium, followed by primary culture for 20 to 24 hours at 31° C. and 150 rpm (OD.sub.610=15 to 20). Next, 1% of the primary culture was inoculated into a 5-L jar fermenter, and 2 to 2.5 L of a seed culture medium was added thereto, followed by secondary culture at 31° C., pH 7.1, 600 rpm and an aeration rate of 1.0 vvm for 21 to 24 hours (OD.sub.610=20 to 40), thereby preparing an inosinic acid seed culture broth. The composition of the seed culture medium used is shown in Table 11 below.

TABLE-US-00011 TABLE 11 Composition Seed culture 4 to 6% glucose, 2 to 4% yeast extract paste, medium for 0.3% (NH.sub.4).sub.2SO.sub.4, 0.2% KH.sub.2PO.sub.4, 0.2% K.sub.2HPO.sub.4, 200 to inosinic acid- 300 ppm adenine, 200 to 300 ppm guanine, 0.15% producing MgSO.sub.4•7H.sub.2O, 10 ppm nicotinic acid, 100 ppm Ca- microorganism pantothenate, 15 ppm cysteine, 1 ppm thiamine HCl, 5 ppm ZnSO.sub.4•7H.sub.2O, 10 ppm MnSO.sub.4•5H.sub.2O, 0.1 ppm biotin, 15 ppm FeSO.sub.4•7H.sub.2O, and 0.01% antifoaming agent for food

[0178] 5-2. Main Fermentation

[0179] In order to examine the ratio of glutamic acid to inosinic acid in the fermentation broth depending on the inoculums of the glutamic acid seed culture broth and the inosinic acid seed culture broth, the glutamic acid seed culture broth and the inosinic acid seed culture broth were inoculated at various ratios and fermented.

[0180] In main fermentation, 14 to 18 L of a fermentation medium was added to a 50-L fermenter and inoculated with 1.2 to 1.8 L of the total seed culture broth at an inoculation ratio of 0.05 to 99.95 (glutamic acid seed culture broth): 99.95 to 0.05 (inosinic acid seed culture broth), followed by mixed fermentation by fed-batch culture for 30 to 90 hours. The fermentation medium composition and fermentation conditions used here are shown in Table 12 below.

TABLE-US-00012 TABLE 12 Fermentation 1 to 3% molasses, 5 to 7% raw sugar, 2 to 3% yeast medium extract paste, 0.6 to 1.2% H.sub.3PO.sub.4, 0.05 to 0.1% composition betaine, 100 to 200 ppm adenine, 50 to 150 ppm guanine, 0.2 to 0.5% MgSO.sub.4•7H.sub.2O, 50 to 100 ppm Ca- pantothenate, 5 to 15 ppm vitamin B3, 5 to 20 ppm thiamine HCl, 0.4 to 0.8% NaOH, 5 to 10 ppm FeSO.sub.4, 10 to 20 ppm MnSO.sub.4, 10 to 20 ppm ZnSO.sub.4, and 0.005% antifoaming agent for food Fermentation Temperature 31 to 32° C., pH 6.5 to 7.5, aeration conditions rate 0.8 to 1.2 vvm, internal pressure 0.6 to 1.0 kg/cm.sup.3, agitation speed 320 to 350 rpm, and dissolved oxygen (DO) concentration 20 to 70%

[0181] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 13 below.

TABLE-US-00013 TABLE 13 Inoculation ratio GA:IMP ratio between seed culture Fermentation in fermentation broths (GA:IMP) time broth 99.95:0.05  33 hours 99.8:1 80:20 42 hours 51.3:1 70:30 48 hours 31.2:1 50:50 60 hours 16.8:1 20:80 83 hours 1.04:1  0.05:99.95 90 hours 0.02:1

[0182] Referring to Table 13 above, it was confirmed that, when the seed culture broths of the microorganisms were inoculated at a ratio of 0.05 to 99.95 (glutamic acid-producing microorganism):99.95 to 0.05 (inosinic acid-producing microorganism), L-glutamic acid and IMP in the fermentation broth were produced at a ratio of 0.02 to 99.8:1.

Example 6. Comparison of Glutamic Acid-Inosinic Acid Fermented Powders Between Fermentation Processes

[0183] 6-1. Production of Glutamic Acid-Inosinic Acid Fermented Powder

[0184] Conventionally, a flavor containing glutamic acid and inosinic acid was produced by individually fermenting a glutamic acid-producing microorganism and an inosinic acid-producing microorganism and then mixing the fermentation broths or dried products thereof at a suitable ratio. To compare the differences in taste between this conventional individual fermentation method and the method based on mixed fermentation of the glutamic acid-producing microorganism and the inosinic acid-producing microorganism, the components of glutamic acid-inosinic acid (GA-IMP) fermented powders obtained by the production methods were compared (see FIG. 3).

[0185] The glutamic acid seed culture broth and inosinic acid seed culture broth used here were prepared in the same manner as in Example 5-1.

{circle around (1)} Individual Fermentation (Preparation Example 7)

[0186] In the method of mixing individual dried products among the conventional methods, each of the glutamic acid seed culture broth and the inosinic acid seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. Next, cells were separated from each fermentation broth, and then decolorization and filtration processes were performed. The filtrates were concentrated and dried to obtain dried products. The obtained dried glutamic acid and isosinic acid products were mixed together so that the ratio of glutamic acid to inosinic acid was 1:1, thereby preparing fermented powder containing glutamic acid and inosinic acid.

{circle around (2)} Mixing of Fermentation Broths after Individual Fermentation (Preparation Example 8)

[0187] In the method of mixing individual fermentation broths among the conventional methods, each of the glutamic acid seed culture broth and the inosinic acid seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. The fermentation broths obtained in the main fermentation were mixed together so that the ratio of glutamic acid to inosinic acid was 1:1. Next, cells were separated from the fermentation broth mixture, and then decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-inosinic acid fermented powder.

{circle around (3)} Mixed Fermentation (Preparation Example 9)

[0188] In mixed fermentation, the glutamic acid seed culture broth and the inosinic acid seed culture broth were inoculated at a ratio of 20:80 in the same manner as in Example 5-2 so that the ratio of glutamic acid to inosinic acid in the fermentation broth was about 1:1 in the same manner as in Preparation Examples 7 and 8, followed by mixed fermentation. Next, cells were separated from the fermentation broth, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain glutamic acid-inosinic acid fermented powder.

[0189] 6-2. Comparison of Components Between Glutamic Acid-Inosinic Acid Fermented Powders

[0190] Component analysis was performed on the glutamic acid-inosinic acid fermented powder obtained through individual fermentation in each of Preparation Examples 7 and 8 and the glutamic acid-inosinic acid fermented powder obtained through mixed fermentation in Preparation Example 9.

[0191] L-glutamic acid and IMP were measured by HPLC analysis (GA—210 nm, UV detector, flow rate 0.9 ml/min; IMP—254 nm, UV detector, flow rate 0.9 ml/min). Organic acid and ions were measured in the same manner as in Example 2-2. The results are shown in Table 14 below.

TABLE-US-00014 TABLE 14 Preparation Preparation Preparation Item Example 7 Example 8 Example 9 GA  22%  31%  37% IMP  20%  31%  36% (GA + IMP)/TS*  42%  62%  73% Total nitrogen 8.6% 9.7% 10.5%  Organic acids 4.0% 3.3% 3.1% Ions 10.4%  8.8% 4.2% Ammonium 4.0% 3.0% 1.7% *(GA + IMP)/TS: Proportion of product relative to total solid content

[0192] 6-3. Sensory Comparison Between Individual Fermentation and Mixed Fermentation

[0193] Sensory evaluation was performed on the glutamic acid-inosinic acid fermented powders obtained through individual fermentation in Preparation Examples 7 and 8 and the glutamic acid-inosinic acid fermented powder obtained through mixed fermentation in Preparation Example 9.

[0194] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 15 below.

TABLE-US-00015 TABLE 15 Preparation Preparation Preparation Example 7 Example 8 Example 9 (individual (individual (mixed Item fermentation) fermentation) fermentation) Initial Umami taste +++ ++++ ++++ Umami persistence + +++ ++++ Kokumi + ++ +++ Salty taste + ++ ++ Sour taste ++ ++ + Bitter taste ++ + − Sweet taste + + ++

[0195] Referring to Table 15 above, due to the difference in components of the glutamic acid-inosinic acid fermented powder as in Table 14 above, the sample prepared by the mixing process after individual fermentation showed a weak umami taste and umami persistence and an increased bitter taste compared to the sample prepared by mixed fermentation. In addition, since the increase in by-products such as organic acids and the increase in ions as shown in Table 14 above also affect the sensory properties of the sample, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after individual fermentation.

Example 7. Mixed Fermentation of Inosinic Acid and Lysine

[0196] 7-1. Seed Culture

[0197] Corynebacterium ammoniagenes NFI545 (KCCM13162P) was used as an inosinic acid-producing microorganism, and Corynebacterium glutamicum NFL21 (KCCM13163P) was used as a lysine-producing microorganism.

[0198] An inosinic acid seed culture broth of the inosinic acid-producing microorganism and a lysine seed culture broth of the lysine-producing microorganism were prepared in the same manner as in Examples 5-1 and 1-1, respectively.

[0199] 7-2. Main Fermentation

[0200] In order to examine the ratio of inosinic acid to lysine in the fermentation broth depending on the inoculums of the inosinic acid seed culture broth and the lysine seed culture broth, the inosinic acid seed culture broth and the lysine seed culture broth were inoculated at various ratios and fermented.

[0201] In main fermentation, 14 to 18 L of a fermentation medium was added to a 50-L fermenter and inoculated with 1.2 to 1.8 L of the total seed culture broth at an inoculation ratio of 0.05 to 99.95 (inosinic acid seed culture broth): 99.95 to 0.05 (lysine seed culture broth), followed by mixed fermentation by fed-batch culture for 45 to 90 hours. The fermentation medium composition and fermentation conditions used here are shown in Table 16 below.

TABLE-US-00016 TABLE 16 Fermentation 1 to 3% molasses, 5 to 7% raw sugar, 2 to 3% yeast medium extract paste, 0.6 to 1.2% H.sub.3PO.sub.4, 0.05 to 0.1% betaine, composition 100 to 200 ppm adenine, 50 to 150 ppm guanine, 0.2 to 0.5% MgSO.sub.4•7H.sub.2O, 50 to 100 ppm Ca-pantothenate, 5 to 15 ppm vitamin B3, 5 to 20 ppm thiamine HCl, 0.4% (NH.sub.4).sub.2SO.sub.4, 0.4 to 0.8% NaOH, 5 to 10 ppm FeSO.sub.4, 10 to 20 ppm MnSO.sub.4, 10 to 20 ppm ZnSO.sub.4, and 0.005% antifoaming agent for food Fermentation Temperature 31 to 32° C., pH 6.5 to 7.5, aeration rate conditions 0.8 to 1.2 vvm, internal pressure 0.6 to 1.0 kg/cm.sup.3, agitation speed 320 to 350 rpm, and dissolved oxygen (DO) concentration 20 to 70%

[0202] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 17 below.

TABLE-US-00017 TABLE 17 Inoculation ratio IMP:LYS ratio between seed culture Fermentation in fermentation broths (IMP:LYS) time ratio 99.95:0.05  90 hours 92.3:1 80:20 73 hours 1.01:1 50:50 61 hours 0.05:1 20:80 48 hours 0.02:1  0.05:99.95 45 hours 0.01:1

[0203] Referring to Table 17 above, it was confirmed that, when the seed culture broths of the microorganisms were inoculated at a ratio of 0.05 to 99.95 (inosinic acid-producing microorganism):99.95 to 0.05 (lysine-producing microorganism), IMP and lysine in the fermentation broth were produced at a ratio of 0.01 to 92:1.

Example 8. Comparison of Inosinic Acid-Lysine Fermented Powders Between Fermentation Processes

[0204] 8-1. Production of Inosinic Acid-Lysine Fermented Powder

[0205] Conventionally, a flavor containing inosinic acid and lysine was produced by individually fermenting an inosinic acid-producing microorganism and a lysine-producing microorganism and then mixing the fermentation broths or dried products thereof at a suitable ratio. To compare the differences in taste between this conventional individual fermentation method and the method based on mixed fermentation of the inosinic acid-producing microorganism and the lysine-producing microorganism, the components of inosinic acid-lysine (IMP-LYS) fermented powders obtained by the production methods were compared (see FIG. 4).

[0206] The inosinic acid seed culture broth and lysine seed culture broth used here were prepared in the same manner as in Example 7-1.

{circle around (1)} Individual Fermentation (Preparation Example 10)

[0207] In the method of mixing individual dried products among the conventional methods, each of the inosinic acid seed culture broth and the lysine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. Next, cells were separated from each fermentation broth, and then decolorization and filtration processes were performed. The filtrates were concentrated and dried to obtain dried products. The obtained dried inosinic acid and lysine products were mixed together so that the ratio of inosinic acid to lysine was 1:1, thereby preparing fermented powder containing inosinic acid and lysine.

{circle around (2)} Mixing of Fermentation Broths after Individual Fermentation (Preparation Example 11)

[0208] In the method of mixing individual fermentation broths among the conventional methods, each of the inosinic acid seed culture broth and the lysine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. The fermentation broths obtained in the main fermentation were mixed together so that the ratio of inosinic acid to lysine was 1:1. Next, cells were separated from the fermentation culture broth, and then decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain inosinic acid-lysine fermented powder.

{circle around (3)}Mixed Fermentation (Preparation Example 12)

[0209] In mixed fermentation, the inosinic acid seed culture broth and the lysine seed culture broth were inoculated at a ratio of 80:20 in the same manner as in Example 7-2 so that the ratio of inosinic acid to lysine in the fermentation broth was about 1:1 in the same manner as in Preparation Examples 10 and 11, followed by mixed fermentation. Next, cells were separated from the fermentation broth, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain inosinic acid-lysine fermented powder.

[0210] 8-2. Comparison of Components Between Lysine-Inosinic Acid Fermented Powders

[0211] Component analysis was performed on the inosinic acid-lysine fermented powder obtained through individual fermentation in each of Preparation Examples 10 and 11 and the inosinic acid-lysine fermented powder obtained through mixed fermentation in Preparation Example 12.

[0212] IMP and L-lysine were measured by HPLC analysis (IMP—254 nm, UV detector, flow rate 0.9 ml/min; LYS—214 nm, UV detector, flow rate 0.8 ml/min). Organic acid and ions were measured in the same manner as in Example 2-2. The results are shown in Table 18 below.

TABLE-US-00018 TABLE 18 Preparation Preparation Preparation Item Example 10 Example 11 Example 12 LYS  26%  32%  38% IMP  24%  30%  33% (LYS + IMP)/TS*  45%  62%  71% Total nitrogen 9.7% 11.3%  12.5%  Organic acids 5.0% 4.1% 3.7% Ions 9.2% 8.2% 5.6% Ammonium 3.9% 3.5% 1.9% *(LYS + IMP)/TS: proportion of product relative to total solid

[0213] Referring to Table 18 above, mixed fermentation (Preparation Example 12) showed a higher LYS+IMP proportion than individual fermentation (Preparation Examples 10 and 11) and showed significant decreases in the contents of organic acids, ions and ammonium.

[0214] 8-3. Sensory Comparison Between Individual Fermentation and Mixed Fermentation

[0215] Sensory evaluation was performed on the inosinic acid-lysine fermented powders obtained through individual fermentation in Preparation Examples 10 and 11 and the inosinic acid-lysine fermented powder obtained through mixed fermentation in Preparation Example 12.

[0216] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 19 below.

TABLE-US-00019 TABLE 19 Preparation Preparation Preparation Example 10 Example 11 Example 12 (individual (individual (mixed Item fermentation) fermentation) fermentation) Initial Umami taste + ++ ++ Umami persistence + +++ ++++ Kokumi + ++ +++ Salty taste + ++ ++ Sour taste ++ ++ + Bitter taste ++ ++ − Sweet taste + + ++

[0217] Referring to Table 19 above, due to the difference in components of the inosinic acid-lysine fermented powder as shown in Table 18 above, the sample prepared by the mixing process after individual fermentation showed weak umami persistence and an increased bitter taste compared to the sample prepared by mixed fermentation. In addition, since the increase in by-products such as organic acids and the increase in ions as shown in Table 18 above also affect the sensory properties of the sample, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after individual fermentation.

Example 9. Mixed Fermentation of Inosinic Acid and Arginine

[0218] 7-1. Seed Culture

[0219] Corynebacterium ammoniagenes NFI545 (KCCM13162P) was used as an inosinic acid-producing microorganism, and Corynebacterium glutamicum NFA40 (KCCM13165P) was used as an arginine-producing microorganism.

[0220] An inosinic acid seed culture broth of the inosinic acid-producing microorganism and an arginine seed culture broth of the arginine-producing microorganism were prepared in the same manner as in Examples 5-1 and 3-1, respectively.

[0221] 9-2. Main Fermentation

[0222] In order to examine the ratio of inosinic acid to arginine in the fermentation broth depending on the inoculums of the inosinic acid seed culture broth and the arginine seed culture broth, the inosinic acid seed culture broth and the arginine seed culture broth were inoculated at various ratios and fermented.

[0223] In main fermentation, 14 to 18 L of a fermentation medium was added to a 50-L fermenter and inoculated with 1.2 to 1.8 L of the total seed culture broth at an inoculation ratio of 65 to 99.95 (inosinic acid seed culture broth): 35 to 0.05 (arginine seed culture broth), followed by mixed fermentation by fed-batch culture for 45 to 80 hours. The fermentation medium composition and fermentation conditions used here are shown in Table 20 below.

TABLE-US-00020 TABLE 20 Fermentation 1 to 3% molasses, 5 to 7% raw sugar, 2 to 3% yeast medium extract paste, 0.6 to 1.2% H.sub.3PO.sub.4, 0.05 to 0.1% composition betaine, 100 to 200 ppm adenine, 50 to 150 ppm guanine, 0.2 to 0.5% MgSO.sub.4•7H.sub.2O, 50 to 100 ppm Ca- pantothenate, 5 to 15 ppm vitamin B3, 5 to 20 ppm thiamine HCl, 0.4% (NH.sub.4).sub.2SO.sub.4, 0.4 to 0.8% NaOH, 5 to 10 ppm FeSO.sub.4, 10 to 20 ppm MnSO.sub.4, 10 to 20 ppm ZnSO.sub.4, and 0.005% antifoaming agent for food Fermentation Temperature 31 to 32° C., pH 6.5 to 7.5, aeration conditions rate 0.8 to 1.2 vvm, internal pressure 0.6 to 1.0 kg/cm.sup.3, agitation speed 320 to 350 rpm, and dissolved oxygen (DO) concentration 20 to 70%

[0224] This mixed fermentation was performed a total of three times, the average value was calculated, and the results are shown in Table 21 below.

TABLE-US-00021 TABLE 21 Inoculation ratio IMP:ARG ratio between seed culture Fermentation in fermentation broths (IMP:ARG) time broth 99.95:0.05  80 hours 95.1:1 80:20 46 hours  5.3:1 70:30 45 hours 2.45:1 65:35 45 hours 1.04:1

[0225] Referring to Table 21 above, it was confirmed that, when the seed culture broths of the microorganisms were inoculated at a ratio of 65 to 99.95 (inosinic acid-producing microorganism):35 to 0.05 (arginine-producing microorganism), IMP and L-arginine in the fermentation broth were produced at a ratio of 1.04 to 95.1:1.

Example 10. Comparison of Inosinic Acid-Arginine Fermented Powders Between Fermentation Processes

[0226] 10-1. Production of Inosinic Acid-Arginine Fermented Powder

[0227] Conventionally, a flavor containing inosinic acid and arginine was produced by individually fermenting an inosinic acid-producing microorganism and an arginine-producing microorganism and then mixing the fermentation broths or dried products thereof at a suitable ratio. To compare the differences in taste between this conventional individual fermentation method and the method based on mixed fermentation of the inosinic acid-producing microorganism and the arginine-producing microorganism, the components of inosinic acid-arginine (IMP-ARG) fermented powders obtained by the production methods were compared (see FIG. 5).

[0228] The inosinic acid seed culture broth and arginine seed culture broth used here were prepared in the same manner as in Example 9-1.

{circle around (1)} Individual Fermentation (Preparation Example 13)

[0229] In the method of mixing individual dried products among the conventional methods, each of the inosinic acid seed culture broth and the arginine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. Next, cells were separated from each fermentation broth, and then decolorization and filtration processes were performed. The filtrates were concentrated and dried to obtain dried products. The obtained dried inosinic acid and arginine products were mixed together so that the ratio of inosinic acid to arginine was 1:1, thereby preparing fermented powder containing inosinic acid and arginine.

{circle around (2)} Mixing of Fermentation Broths after Individual Fermentation (Preparation Example 14)

[0230] In the method of mixing individual fermentation broths among the conventional methods, each of the inosinic acid seed culture broth and the arginine seed culture broth was transferred into a 50-L fermenter and then individually subjected to main fermentation. The fermentation broths obtained in the main fermentation were mixed together so that the ratio of inosinic acid to arginine was 1:1. Next, cells were separated from the fermentation broth mixture, and then decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain inosinic acid-arginine fermented powder.

{circle around (3)} Mixed Fermentation (Preparation Example 15)

[0231] In mixed fermentation, the inosinic acid seed culture broth and the arginine seed culture broth were inoculated at a ratio of 65:35 in the same manner as in Example 9-2 so that the ratio of inosinic acid to arginine in the fermentation broth was about 1:1 in the same manner as in Preparation Examples 13 and 14, followed by mixed fermentation. Next, cells were separated from the fermentation broth, and decolorization and filtration processes were performed. The filtrate was concentrated and dried to obtain inosinic acid-arginine fermented powder.

[0232] 10-2. Comparison of Components Between Inosinic Acid-Arginine Fermented Powders

[0233] Component analysis was performed on the inosinic acid-arginine fermented powder obtained through individual fermentation in each of Preparation Examples 13 and 14 and the inosinic acid-arginine fermented powder obtained through mixed fermentation in Preparation Example 15.

[0234] L-arginine and IMP were measured by HPLC analysis (ARG—195 nm, UV detector, flow rate 1 ml/min; IMP—254 nm, UV detector, flow rate 0.9 ml/min). Organic acid and ions were measured in the same manner as in Example 2-2. The results are shown in Table 22 below.

TABLE-US-00022 TABLE 22 Preparation Preparation Preparation Item Example 13 Example 14 Example 15 ARG  24%  31%  34% IMP  24%  31%  33% (ARG + IMP)/TS*  48%  62%  76% Total nitrogen 9.7% 11.3%  14.1%  Organic acids 4.7% 3.8% 2.5% Ions 7.5% 6.2% 3.8% Ammonium 3.1% 2.5% 0.8% *(ARG + IMP)/TS: Proportion of product relative to total solid content

[0235] 10-3. Sensory Comparison Between Individual Fermentation and Mixed Fermentation

[0236] Sensory evaluation was performed on the inosinic acid-arginine fermented powders obtained through individual fermentation in Preparation Examples 13 and 14 and the inosinic acid-arginine fermented powder obtained through mixed fermentation in Preparation Example 15.

[0237] Sensory evaluation was performed in the same manner as in Example 2-3. The results are shown in Table 23 below.

TABLE-US-00023 TABLE 23 Preparation Preparation Preparation Example 13 Example 14 Example 15 (individual (individual (mixed Item fermentation) fermentation) fermentation) Initial Umami taste + ++ ++ Umami persistence + +++ ++++ Kokumi + ++ +++ Salty taste + ++ ++ Sour taste ++ ++ + Bitter taste ++ ++ − Sweet taste − − −

[0238] Referring to Table 23 above, due to the difference in components of the inosinic acid-arginine fermented powder as shown in Table 22 above, the sample prepared by the mixing process after individual fermentation showed weak umami persistence and an increased bitter taste compared to the sample prepared by mixed fermentation. In addition, since the increase in by-products such as organic acids and the increase in ions as shown in Table 22 above also affect the sensory properties of the sample, the mixed fermentation process is more effective in terms of taste or process simplification than mixing after individual fermentation.

[0239] So far, the present invention has been described with reference to the preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will appreciate that the present invention may be embodied in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view, not from a restrictive point of view. The scope of the present invention is defined by the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the scope of the present invention.

ACCESSION NUMBERS

[0240] Depository authority: Korean Culture Center of Microorganisms (KCCM)

[0241] Accession number: KCCM13162P

[0242] Deposit date: Apr. 21, 2022

[0243] Depository authority: Korean Culture Center of Microorganisms (KCCM)

[0244] Accession number: KCCM13163P

[0245] Deposit date: Apr. 21, 2022

[0246] Depository authority: Korean Culture Center of Microorganisms (KCCM)

[0247] Accession number: KCCM13164P

[0248] Deposit date: Apr. 21, 2022

[0249] Depository authority: Korean Culture Center of Microorganisms (KCCM)

[0250] Accession number: KCCM13165P

[0251] Deposit date: Apr. 21, 2022