METHOD FOR PREPARING FERMENTED COMPOSITION WITH IMPROVED ODOR USING YEAST

Abstract

The present disclosure relates to a method for preparing a fermented composition, and more specifically, to a method for preparing a fermented composition with improved odor, which comprises preparing grain flour; performing primary fermentation of the grain flour using yeast; performing secondary fermentation of the primary fermented product using a strain of the genus Bacillus; and obtaining the secondary fermented product. The fermented composition of the present disclosure has a high content of a peptide with a low molecular weight, and thus enables the increase of digestibility and absorption rate of proteins during ingestion while also improving the peculiar odor of a fermented product to enhance its palatability.

Claims

1: A method for preparing a fermented composition with improved odor, comprising: preparing grain flour; performing primary fermentation of the grain flour using yeast; performing secondary fermentation of the primary fermented product using a strain of the genus Bacillus; and obtaining the secondary fermented product.

2: The method according to claim 1, wherein the yeast produces -galactosidase, protease, and phytase.

3: The method according to claim 1, wherein the yeast is Saccharomyces cerevisiae deposited under Accession Number KCCM12123P or Accession Number KCCM12124P.

4: The method according to claim 1, wherein, in the secondary fermented product, a peptide with a molecular weight of 30 kDa or less is contained in an amount of 40% to 100%.

5: The method according to claim 1, wherein the grain flour comprises soybean meal or corn gluten.

6: The method according to claim 1, wherein the performing of the primary fermentation of the grain flour comprises adding -amylase or glucoamylase.

7: The method according to claim 1, wherein the grain flour undergoes moisture content adjustment and then heat treatment.

8: The method according to claim 7, wherein the adjusted moisture content is in a range of 30% to 60%.

9: The method according to claim 1, wherein the strain of the genus Bacillus is at least one strain selected from the group consisting of Bacillus subtilis, Bacillus licheniformis, Bacillus toyoi, Bacillus coagulans, Bacillus polyfermenticus, and Bacillus amyloliquefaciens.

10: The method according to claim 1, wherein the strain of the genus Bacillus is Bacillus amyloliquefaciens deposited under Accession Number KCCM114371P.

11: Yeast for improving the odor of a fermented product of Bacillus which produces -galactosidase, protease, and phytase.

12: The yeast according to claim 11, wherein the yeast is Saccharomyces cerevisiae deposited under Accession Number KCCM12123P or Accession Number KCCM12124P.

13: A composition for grain fermentation comprising the yeast of claim 12.

14: A feed composition comprising the yeast of claim 12.

15: A fermented composition prepared by a method according to claim 1.

16: A feed composition comprising the fermented composition of claim 15.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0066] FIG. 1 shows an image illustrating the measurement results of saccharide components in a group of grain raw materials, a group of mixed grains with Bacillus fermentation alone, a group of mixed grains with yeast fermentation alone, and a group of mixed grains with combined fermentation by yeast and Bacillus, in which (1) represents standard materials: stachyose, raffinose, sucrose, and glucose (from the bottom); (2) represents raw materials of soybean meal; (3) represents raw materials of corn gluten; (4) represents mixed grains (soybean meal+corn gluten) with yeast fermentation alone; (5) represents mixed grains (soybean meal+corn gluten) with yeast (CJN1697) fermentation alone; (6) represents mixed grains (soybean meal+corn gluten) with combined fermentation by yeast (CJN1697) and Bacillus; (7) represents mixed grains (soybean meal+corn gluten) with combined fermentation by yeast (CJN2343) and Bacillus; and (8) represents mixed grains (soybean meal+corn gluten) with combined fermentation by yeast (Angest) and Bacillus.

[0067] FIG. 2 shows an image illustrating the analysis results of protein components for each of the following groups: the group of grain raw materials (soybean meal, corn gluten, and mixed grain raw materials); the group of the grain raw materials with Bacillus fermentation alone; the group of the grain raw materials with yeast fermentation alone; and the group of the grain raw materials with combined fermentation by yeast and Bacillus.

[0068] FIG. 3 shows a chart illustrating the results of the phylogenetic analysis of CJN1697 strain.

[0069] FIG. 4 shows a chart illustrating the results of the phylogenetic analysis of CJN2343 strain.

DETAILED DESCRIPTION OF THE INVENTION

[0070] Hereinafter, the present disclosure will be described in detail through exemplary embodiments. However, these exemplary embodiments are for illustrative purposes only and are not intended to limit the scope of the present disclosure.

Example 1: Screening of Yeast Strains

[0071] Among the yeast strains, those strains with excellent abilities of producing -galactosidase, protease, and phytase were selected. To confirm the ability of producing -galactosidase, the X-gal agar medium (NaCl (0.5%), peptone (1%), raffinose (1%), agar (1.5%), and X-gal (0.5%)) was prepared. Additionally, to confirm the ability of producing protease, the YM agar medium (powdered skim milk (2%), yeast extract (0.3%), malt extract (0.3%), peptone (1%), and agar (1.5%)) were prepared, and to measure the phytase activity, the medium was prepared by adding phytin to the above medium.

[0072] Each yeast strain was cultured in the YPD medium (glucose (2%), yeast extract (0.8%), and soy peptone (0.2%)) at 30 C. for 12 hours and thereby the Saccharomyces cerevisiae strain was prepared. The prepared yeast culture (5 L) was added dropwise to each agar medium, cultured at 30 C. for about 24 hours, and the abilities of producing -galactosidase, protease, and phytase were measured by the clear zones generated where each drop of the yeast culture was placed.

[0073] The presence of -galactosidase was confirmed through the X-gal agar medium, and the abilities of producing protease and phytase were examined by measuring and comparing the size of each colony and the clear zone generated around the colony (clear zone size/colony size) (Table 1). In the case of strains where no clear zone generation was observed due to the absence of protease activity, these strains were indicated as 0. As a result, among the about 100 Saccharomyces cerevisiae strains, 14 strains were shown to have the -galactosidase activity, and among the 14 strains, two strains (i.e., CJN1697 and CJN2343) were ultimately selected by excluding those strains where the protease activity necessary for grain fermentation is not present at all.

TABLE-US-00001 TABLE 1 Ratio (Clear Zone/Colony Presence of - Size) Strain No. Galactosidase Protease Phytase CJN1023 X 1.11 1.58 CJN1025 X 1.51 1.11 CJN1102 X 1.07 1.24 CJN1103 X 1.19 1.29 CJN1105 X 1.22 1.48 CJN1108 X 1.11 1.25 CJN1110 X 1.11 1.33 CJN1145 X 1.03 1.32 CJN1146 X 1.02 1.43 CJN1147 X 1.00 1.58 CJN1148 X 1.00 1.32 CJN1292 X 1.21 1.42 CJN1293 X 1.00 1.52 CJN1294 X 1.13 1.31 CJN1295 X 1.14 1.59 CJN1297 X 1.10 1.38 CJN1298 X 1.19 1.71 CJN1334 X 1.23 1.14 CJN1336 X 1.00 1.24 CJN1437 X 1.00 1.33 CJN1439 X 1.00 1.11 CJN1440 X 1.12 1.65 CJN1441 X 1.02 1.43 CJN1442 X 1.10 1.66 CJN1443 X 1.09 1.30 CJN1697 O 1.02 1.50 CJN1837 X 1.00 1.29 CJN1838 X 1.06 1.41 CJN1839 X 1.04 1.41 CJN1840 X 1.03 1.37 CJN1841 X 1.02 1.41 CJN1842 X 1.09 1.39 CJN1843 X 1.09 1.26 CJN1844 X 1.06 1.37 CJN1845 X 1.06 1.24 CJN1846 X 1.04 1.37 CJN1907 X 1.55 1.12 CJN1973 X 1.05 1.23 CJN1974 X 0.00 1.29 CJN1975 X 0.00 1.19 CJN1976 X 0.00 1.23 CJN2022 X 0.00 1.33 CJN2023 X 0.00 1.20 CJN2024 X 0.00 1.32 CJN2025 X 0.00 1.35 CJN2026 X 0.00 1.29 CJN2027 X 0.00 1.36 CJN2028 X 0.00 1.29 CJN2029 X 1.05 1.24 CJN2030 X 0.00 1.14 CJN2031 X 1.03 1.57 CJN2067 X 1.10 1.45 CJN2145 X 1.09 1.42 CJN2147 X 1.03 1.22 CJN2150 X 1.03 1.38 CJN2225 X 1.03 1.32 CJN2229 X 1.03 1.20 CJN2343 O 1.13 1.25 CJN2347 X 1.11 1.33 CJN2350 X 1.08 1.34 CJN2387 X 1.07 1.41 CJN2389 X 1.09 1.42 CJN2390 X 1.08 1.39 CJN2391 X 1.05 1.37 CJN2393 X 1.11 1.32 CJN2394 X 1.10 1.41 CJN2395 X 1.10 1.39 CJN2560 O 0.00 1.09 CJN2562 O 0.00 1.06 CJN2563 O 0.00 1.03 CJN2564 O 0.00 1.13 CJN2565 O 0.00 1.18 CJN2597 O 0.00 1.17 CJN2600 O 0.00 1.23 CJN2601 O 0.00 1.16 CJN2602 O 0.00 1.10 CJN2603 X 0.00 1.19 CJN2604 O 0.00 1.10 CJN2605 O 0.00 1.13 CJN2606 O 0.00 1.13 CJN2642 X 0.00 1.03 CJN2643 X 0.00 1.19 CJN2644 X 0.00 1.11 CJN2645 X 0.00 1.15 CJN2646 X 0.00 0.00 CJN2647 X 0.00 1.26 CJN2648 X 0.00 1.17 CJN2649 X 0.00 1.13 CJN2650 X 0.00 1.00 CJN2651 X 0.00 1.12 CJN2672 X 0.00 1.09 CJN2673 X 0.00 1.06 CJN2674 X 0.00 1.09 CJN2675 X 0.00 1.12 CJN2676 X 0.00 1.10 CJN2677 X 0.00 1.09 CJN2678 X 0.00 1.14 CJN2679 X 0.00 1.10 CJN2680 X 0.00 1.11 CJN2681 X 0.00 1.18 CJN2682 X 0.00 1.11 CJN2683 X 0.00 1.09 CJN2684 X 0.00 1.10 CJN2685 X 0.00 1.06 CJN2686 X 0.00 1.17 CJN2687 X 0.00 1.12 CJN2688 X 0.00 1.20 CJN2689 X 0.00 1.16 CJN2690 X 0.00 1.03

Example 2: Preparation of Fermented Composition

[0074] 2-1. Method of Moisture Treatment and Heat Treatment of Grain Flour

[0075] Soybean meal flour and corn gluten flour were each prepared. Water was added to the soybean meal to adjust the moisture content of the soybean meal to about 45% based on the weight of the soybean meal and the mixture was subjected to heat treatment at 100 C. for 30 minutes. For the corn gluten flour, phosphoric acid was added in an amount of 1.5 wt % based on the weight of the corn gluten, and then water was added to adjust the moisture content of the corn gluten to about 43%, and the mixture was subjected to heat treatment at 100 C. for 30 minutes. Sodium hydroxide (NaOH) in an amount of 2.4 wt % based on the weight of the corn gluten was added to the corn gluten, which has undergone heat treatment, and then water was added to adjust the moisture content of the corn gluten to about 45%.

[0076] 2-2. Method of Enzyme Treatment and Preparation of Group with Yeast Fermentation

[0077] The soybean meal flour and corn gluten flour prepared by the method of Example 2-1 were mixed at the same weight ratio to prepare mixed grain flour. Then, glucoamylase (0.5 wt %) was added to the mixed grain flour and inoculated with each culture of three kinds of Saccharomyces cerevisiae (CJN1697, CJN2343, and commercial bread yeast (purchased from Angel Yeast Co., Ltd.)) in an amount of 10 wt % and mixed, and this was allowed to ferment anaerobically at 30 C. for 6 hours, and thereby groups with yeast fermentation were prepared.

[0078] 2-3. Preparation Method of Group with Yeast and Bacillus Fermentation

[0079] The group of yeast fermentation prepared by the method of Example 2-2 was further subjected to Bacillus fermentation. More specifically, as described in Example 2-2, each fermentation group, in which fermentation was performed using the three kinds of Saccharomyces cerevisiae (CJN1697, CJN2343, and commercial bread yeast), and 6 hours after the yeast inoculation, was inoculated with the culture of Bacillus amyloliquefaciens (KCCM11471P) in an amount of 10 wt %, and then aerobic fermentation was performed in a thermo-hygrostat (temperature: 37 C., humidity: 95%) for 24 hours.

[0080] 2-4. Measurement of Components in Each Experimental Group

[0081] For each experimental group prepared by the methods of Examples 2-2 and 2-3, the moisture content, viable cell count of Bacillus, viable cell count of yeast, and protein content were measured according to time. The protein content was measured by a Kjeldahl apparatus after drying and the fermented product was pulverized. The measured results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Increased Level of Protein Protein Moisture Content Content Content Bacillus Yeast (Dry (%) Sample Time (hr) (%) (CFU/g) (CFU/g) Weight, %) CP Group with Yeast 0 1.9 10.sup.7 61.90 1.15 Fermentation 6 50.57 7.5 10.sup.7 63.03 2.29 (CJN1697) 22 35.85 6.1 10.sup.8 67.00 6.26 26 33.66 7.3 10.sup.8 67.31 6.56 30 32.78 1.4 10.sup.4 6.9 10.sup.8 66.26 5.51 Group 1 with Yeast 0 1.9 10.sup.7 61.27 0.52 and Bacillus 6 50.66 1.1 10.sup.7 8.4 10.sup.7 63.63 2.89 Fermentation 22 44.21 5.9 10.sup.9 3.8 10.sup.8 69.70 8.95 (CJN1697 + 26 39.50 9.0 10.sup.9 5.4 10.sup.8 69.90 9.15 Bacillus 30 35.61 8.6 10.sup.9 3.9 10.sup.8 70.94 10.20 amyloliquefaciens ) Group 2 with Yeast 0 2.1 10.sup.7 62.32 1.57 and Bacillus 6 50.72 1.5 10.sup.7 8.3 10.sup.7 64.51 3.76 Fermentation 22 46.00 3.1 10.sup.9 4.2 10.sup.8 68.89 8.14 (CJN2343 + 26 38.46 6.2 10.sup.9 4.5 10.sup.8 70.23 9.48 Bacillus 30 34.06 9.0 10.sup.9 4.7 10.sup.8 70.49 9.74 amyloliquefaciens ) Group 3 with Yeast 0 2.3 10.sup.7 61.40 0.66 and Bacillus 6 50.53 1.1 10.sup.7 6.9 10.sup.7 64.14 3.39 Fermentation 22 48.79 1.9 10.sup.9 3.7 10.sup.8 68.51 7.77 (Commercial 26 42.22 7.3 10.sup.9 3.2 10.sup.8 69.72 8.97 Bread Yeast + 30 38.46 .sup.1.5 10.sup.10 2.3 10.sup.8 70.86 10.12 Bacillus amyloliquefaciens )

[0082] As a result, in all of the experimental groups in which yeast fermentation was performed, the amount of live yeast was increased from 10 CFU/g to 10.sup.8 CFU/g. Additionally, in all of the experimental groups in which Bacillus fermentation was performed, the amount of live Bacillus was increased from 10.sup.9 CFU/g to 10.sup.10 CFU/g. Additionally, it was confirmed that the amount of proteins in the composition increased through yeast fermentation, and it was confirmed that the addition of the Bacillus fermentation after the yeast fermentation was more effective in increasing the protein amount.

[0083] Accordingly, that is, when the Bacillus fermentation is performed on the grain raw materials after the yeast fermentation, the protein content of the grain raw materials can be further increased by Bacillus without inhibiting the growth of yeast.

Example 3: Confirmation of Degree of Oligosaccharide Decomposition by Fermentation

[0084] Two types of groups of grain raw materials (a group of soybean meal raw materials and a group of corn gluten raw materials), a group of mixed grains (soybean meal+corn gluten) with Bacillus fermentation alone, a group of mixed grains (soybean meal+corn gluten) with yeast fermentation alone, and a group of mixed grains (soybean meal+corn gluten) with combined fermentation by yeast and Bacillus were prepared, and saccharide components were analyzed for each group.

[0085] The group of soybean meal raw materials (FIG. 1 (2)) and the group of corn gluten raw materials (FIG. 1 (3)), which were not pretreated, were prepared. The saccharide component of each group was quantitatively analyzed via thin layer chromatography (TLC). 25 mL of distilled water was added to 1 g of each sample, and the mixture was heated in boiling water for 15 to 20 minutes and extracted by shaking at 37 C. for 2 hours. The extract was centrifuged and the supernatant was recovered and used as a TLC sample. 2 L of the supernatant was spotted on a silica gel TLC plate, dried to a constant level, and developed for 3 hours in the developing solution. After the development was completed, the oligosaccharide and monosaccharide spots were confirmed through color development and drying processes.

[0086] The group of mixed grains with Bacillus fermentation alone (FIG. 1 (4)) was prepared by mixing the soybean meal flour and the corn gluten flour prepared by the method of Example 2-1 at the same weight ratio, inoculating with the culture of Bacillus amyloliquefaciens in an amount of 10 wt % to the mixture, followed by performing aerobic fermentation in a thermo-hygrostat (temperature: 37 C., humidity: 95%) for 24 hours.

[0087] The group of mixed grains with yeast fermentation alone (FIG. 1 (5)) was prepared by the method of Example 2-2. For the group of mixed grains with combined fermentation by yeast and Bacillus, 3 groups (FIG. 1 (6) (CJN1697+Bacillus), FIG. 1 (7) CJN2343+Bacillus, and FIG. 1 (8) bread yeast+Bacillus) were prepared using three kinds of yeast.

[0088] The saccharide component of each fermentation group was determined in the same manner for the saccharide component measurement in the group of soybean meal raw materials and the group of corn gluten raw materials. For each fermentation group, 25 mL of distilled water was added to 1 g of each sample, and the mixture was heated in boiling water for 15 to 20 minutes and extracted by shaking at 37 C. for 2 hours. The extract was centrifuged and the supernatant was recovered and used as a TLC sample.

[0089] In FIG. 1, lane (1) represents saccharide component markers of stachyose, raffinose, sucrose, and glucose, in the order of from bottom to top; lane (2) represents the group of soybean meal raw materials; lane (3) represents the group of corn gluten raw materials; lane (4) represents mixed grains (soybean meal+corn gluten) with yeast fermentation alone; lane (5) represents mixed grains (soybean meal+corn gluten) with yeast (CJN1697) fermentation alone; each of lanes (6) to (8) represents the group of mixed grains (soybean meal+corn gluten) with combined fermentation by yeast and Bacillus ((6) CJN1697+Bacillus, (7) CJN2343+Bacillus, and (8) bread yeast+Bacillus).

[0090] Referring to FIG. 1, in the group of single fermentation using Bacillus (FIG. 1 (4)), it was confirmed that the saccharide component contained in the soybean meal was not completely decomposed because the microorganism could not sufficiently utilize the saccharide component during the fermentation process, and thus oligosaccharides were included in the fermented product. Meanwhile, in the group where fermentation was performed using yeast (FIG. 1 (5)), it was confirmed that the oligosaccharides were decomposed by yeast and the microorganism had sufficiently utilized the decomposed saccharide component, thus resulting in a low content of oligosaccharides.

[0091] Additionally, in the fermentation groups where CJN1697 and CJN2343 yeast strains were used (lanes (6) and (7)), no specific oligosaccharide spot was observed in the fermented product due to the -galactosidase activity of the enzyme, whereas in the fermentation group where commercial bread yeast (Angest) was used (lane (8)), the -galactosidase activity of the bread yeast was low and thus a specific oligosaccharide spot was observed.

[0092] Accordingly, the fermented composition prepared using yeast is characterized in that oligosaccharides are decomposed within the composition, and thus the fermented composition does not require any individual digestive enzyme for their decomposition when ingested by an animal through feed, thus making the digestion of feed easier. In particular, it was confirmed that it is more effective to use a yeast strain of CJN1697 or CJN2343.

Example 4: Confirmation of Degree of Protein Decomposition by Fermentation

[0093] In the same manner as in Example 3, groups of grain raw materials (soybean meal, corn gluten, and mixed grain raw materials), a group of grain raw materials with Bacillus fermentation alone, a group of grain raw materials with yeast fermentation alone, and a group of grain raw materials with combined fermentation by yeast and Bacillus were prepared, respectively, and analysis of the protein components in each group was attempted.

[0094] As the group of grain raw materials, a total of three kinds of groups (i.e., a group of soybean meal raw materials (group 2), a group of corn gluten raw materials (group 3), and a group of mixed grains in which soybean meal and corn gluten were mixed at the same weight ratio (group 4) were prepared. The protein molecular weight pattern of each raw material was confirmed by SDS-PAGE. 100 mg of each sample was mixed with 5 mL of an 8 M urea solution, and the mixture was sonicated for extraction and centrifuged, and the supernatant was recovered. In each supernatant, protein content was quantitated using bicinchoninic acid and confirmed by SDS-PAGE by loading a certain amount of each protein sample.

[0095] The groups of grain raw materials with Bacillus fermentation alone (groups 5 to 8) were prepared by inoculating a flour mixture, in which the soybean meal flour and corn gluten flour prepared by the method of Example 2-1 were mixed at the same weight ratio, with the culture of Bacillus amyloliquefaciens in an amount of 10 wt %, followed by performing aerobic fermentation in a thermo-hygrostat (temperature: 37 C., humidity: 95%) for 24 hours. The fermentation time (0 hours, 16 hours, 20 hours, and 24 hours) for each group is shown in Table 3 below.

[0096] The groups of grain raw materials with yeast fermentation alone (groups 9 to 12) were prepared by the method of Example 2-1, and the groups of grain raw materials with combined fermentation by yeast and Bacillus (groups 13 to 24) were prepared by the method of Example 2-3 using three kinds of yeast. The strains used in the fermentation of each group and the fermentation time of each group are shown in Table 3 below. In the groups with combined fermentation where Bacillus fermentation proceeded after yeast fermentation, Bacillus fermentation proceeded 6 hours after yeast fermentation.

[0097] The protein decomposition level of each fermentation group was confirmed by SDS-PAGE. The samples were pretreated in the same manner as in confirming the distribution level of molecular weight of proteins in groups of grain raw materials. The protein molecular weight pattern of each raw material was confirmed by SDS-PAGE. 100 mg of each sample was mixed with an 8 M urea solution, and the mixture was sonicated for extraction and centrifuged, and the supernatant was recovered. The protein content was quantitated using bicinchoninic acid and confirmed by SDS-PAGE by loading a certain amount of each protein sample. The results are shown in FIG. 2.

TABLE-US-00003 TABLE 3 Total Fermentation Time (Yeast Fermentation Time + Bacillus Experimental Grain Raw Yeast Bacillus Fermentation group Material Fermentation Fermentation Time) Group 1 Group 2 Soybean Meal Group 3 Corn Gluten Group 4 Mixed Grain Group 5 Mixed Grain B. amyloliquefaciens 0 Group 6 Mixed Grain B. amyloliquefaciens 16 Group 7 Mixed Grain B. amyloliquefaciens 20 Group 8 Mixed Grain B. amyloliquefaciens 24 Group 9 Mixed Grain CJN1697 6 Group 10 Mixed Grain CJN1697 22 Group 11 Mixed Grain CJN1697 26 Group 12 Mixed Grain CJN1697 30 Group 13 Mixed Grain CJN1697 B. amyloliquefaciens 6 (6 + 0) Group 14 Mixed Grain CJN1697 B. amyloliquefaciens 22 (6 + 16) Group 15 Mixed Grain CJN1697 B. amyloliquefaciens 26 (6 + 20) Group 16 Mixed Grain CJN1697 B. amyloliquefaciens 30 (6 + 24) Group 17 Mixed Grain CJN2343 B. amyloliquefaciens 6 (6 + 0) Group 18 Mixed Grain CJN2343 B. amyloliquefaciens 22 (6 + 16) Group 19 Mixed Grain CJN2343 B. amyloliquefaciens 26 (6 + 20) Group 20 Mixed Grain CJN2343 B. amyloliquefaciens 30 (6 + 24) Group 21 Mixed Grain Bread Yeast B. amyloliquefaciens 6 (6 + 0) Group 22 Mixed Grain Bread Yeast B. amyloliquefaciens 22 (6 + 16) Group 23 Mixed Grain Bread Yeast B. amyloliquefaciens 26 (6 + 20) Group 24 Mixed Grain Bread Yeast B. amyloliquefaciens 30 (6 + 24)

[0098] In Table 3 above, the time of the sequential fermentation groups by yeast and Bacillus means the total fermentation time, which is equal to the sum of the fermentation time by Bacillus and the yeast fermentation time (i.e., 6 hours).

[0099] FIG. 2 shows an image illustrating the SDS-PAGE results of the supernatant proteins in groups 1 to 24.

[0100] Referring to Table 3 and FIG. 2, Bacillus can produce protease and thus can decompose proteins into low-molecular weight peptides during the fermentation process using the protease. Therefore, in the combined fermentation groups by yeast and Bacillus, it was confirmed that the proteins of soybean meal and corn gluten were decomposed. Meanwhile, since yeast cannot produce protease at all, proteins cannot be decomposed at all in the group with yeast fermentation alone, and thus the protein patterns of raw materials were indicated as they were. That is, since the composition which underwent Bacillus fermentation after yeast fermentation contains low-molecular weight peptides, the composition can improve the protein absorption rate of feed.

[0101] To more specifically measure the content of low-molecular weight peptides in the fermented product, the distribution according to the molecular weight of low-molecular weight peptides was measured using the gel permeation chromatography (GPC) method.

[0102] GPC is a method to confirm retention time (RT) by analyzing standard proteins having different molecular weights, and to measure protein distribution of analytes according to molecular weight using molecular weight and a standard curve of RT. To confirm the level of protein decomposition of the raw materials due to fermentation, the protein distribution in the fermented product was analyzed by the GPC method.

[0103] GPC analytes were pretreated in the same manner as in the SDS-PAGE method. 100 mg of each sample was suspended in 5 mL of an 8 M urea solvent, and the mixture was sonicated for extraction and centrifuged, and the recovered supernatant was filtered with a syringe filter and used as an analyte for GPC analysis. As the analyte, each of the fermented products from group 4 (mixed raw materials: soybean meal+corn gluten), group 8 (Bacillus fermentation alone), group 12 (yeast fermentation alone), and groups 16, 20, and 24 (combined fermentation by Bacillus+yeast) was used. The results of GPC analysis are shown in Table 4 below.

TABLE-US-00004 TABLE 4 Mixed Bacillus Yeast Molecular Raw Control Control CJN1697 + B CJN2343 + B AngelY + B Weight Material 24 Hr 30 Hr 30 Hr 30 Hr 30 Hr (kDa) (Group 4) (Group 8) (Group 12) (Group 16) (Group 20) (Group 24) >75 60.11 16.38 58.36 10.51 10.36 9.50 30 to 75 22.89 11.70 24.17 5.74 6.12 5.34 10 to 30 7.37 21.91 7.14 16.51 17.09 16.33 5 to 10 2.78 16.20 2.72 17.93 17.90 17.75 <5 6.86 33.81 7.61 49.31 48.53 51.07 Total 100 100 100 100 100 100

[0104] Referring to Table 4, the raw material contained more than 82% of polymer peptides of 30 kDa or greater. In the case of Bacillus fermentation alone, the content of low-molecular weight peptides of 30 kDa or less was about 71%, whereas in the case of the combined fermentation by Bacillus and yeast, the content of low-molecular weight peptides of 30 kDa or less was about 83%, thus showing a significant increase in the content of low-molecular weight peptides. Additionally, when the fermented product was obtained by Bacillus and yeast, the content of low-molecular weight peptides of 10 kDa or less within the fermented product was in a range of about 66% to about 69%, thus showing an increase of about 40% compared to the content of low-molecular weight peptides of 10 kDa or less in the product fermented by Bacillus fermentation alone. That is, in the case of fermentation by Bacillus and yeast, the protein decomposition efficiency was increased and the content of low-molecular weight peptides was increased in the fermented product. Therefore, it can be seen that the digestion and absorption rate can be significantly improved when the product fermented by Bacillus and yeast is used as a raw material for food or feed.

Example 5: Comparison Between Simultaneous Fermentation or Sequential Fermentation of Yeast and Bacillus

[0105] The viable cell counts and the amount of protein increase were measured and compared between a case where yeast fermentation and Bacillus fermentation are performed simultaneously and a case where yeast fermentation and Bacillus fermentation are performed sequentially.

[0106] The group with yeast fermentation was prepared by the method of Example 2-2 and the group with sequential fermentation by yeast and Bacillus was prepared by the method of Example 2-3. The group with simultaneous fermentation by yeast and Bacillus was prepared by mixing the soybean meal flour and the corn gluten flour prepared by the method of Example 2-1 at the same weight ratio, adding glucoamylase (0.5%) thereto, and inoculating simultaneously with yeast and Bacillus, followed by performing aerobic fermentation. The group with sequential fermentation by yeast and Bacillus was prepared by performing Bacillus fermentation 6 hours after yeast fermentation. The moisture content, the viable cell count of Bacillus, the viable cell count of yeast, and the amount of proteins were measured according to time in each group, and the results are shown in Table 5 below.

TABLE-US-00005 TABLE 5 Increased Protein Level of Content Protein Moisture (Dry Content Time Content Bacillus Yeast Weight, (%) Sample (hr) (%) (CFU/g) (CFU/g) %) CP Bacillus Control 0 49.6 5.5 10.sup.7 61.9 2.3 16 44.6 9.6 10.sup.9 69.2 9.5 20 42.4 .sup.1.3 10.sup.10 69.3 9.6 24 38.5 9.6 10.sup.9 69.6 9.9 Group with Yeast 0 49.1 1.5 10.sup.7 61.0 1.3 Fermentation 16 48.5 3.7 10.sup.8 67.1 7.5 (CJN1697) 20 48.8 4.4 10.sup.8 68.9 9.2 24 48.4 4.1 10.sup.8 67.9 8.2 Group with 0 49.4 4.8 10.sup.7 1.7 10.sup.7 60.9 1.2 Simultaneous 16 41.5 .sup.1.2 10.sup.10 3.1 10.sup.7 68.0 8.4 Fermentation by 20 39.4 .sup.1.3 10.sup.10 1.9 10.sup.7 69.9 10.2 Yeast and Bacillus 24 35.8 .sup.1.1 10.sup.10 1.6 10.sup.7 69.1 9.4 (CJN1697 + Bacillus amyloliquefaciens ) Group with 0 2.8 10.sup.7 60.1 0.4 Sequential 6 (6 + 49.7 6.4 10.sup.7 3.8 10.sup.7 62.4 2.7 Fermentation by 0) Yeast and Bacillus 22 (6 + 45.1 6.5 10.sup.9 1.9 10.sup.8 70.8 11.1 (CJN1697 + 16) Bacillus amyloliquefaciens ) 26 (6 + 43.3 6.9 10.sup.9 2.2 10.sup.8 715 11.9 20) 30 (6 + 40.9 .sup.1.1 10.sup.10 1.3 10.sup.8 70.7 11.0 24)

[0107] In Table 5 above, the time of the sequential fermentation groups by yeast and Bacillus means the total fermentation time which is equal to the sum of the fermentation time by Bacillus and the yeast fermentation time (i.e., 6 hours).

[0108] Referring to Table 5, when Bacillus fermentation was performed following yeast fermentation, both the viable cell count of Bacillus and the viable cell count of yeast increased in proportion to their fermentation time. However, it was confirmed that when yeast and Bacillus were simultaneously inoculated and fermented together, the viable cell count of Bacillus increased in proportion to its fermentation time, but the viable cell count of yeast remained at a level of 10.sup.7 CFU/g. That is, when yeast and Bacillus were simultaneously inoculated and fermented together, yeast did not affect the growth of Bacillus, whereas Bacillus inhibited the growth of yeast.

[0109] Accordingly, it was presumed that the protease of Bacillus reduces the population of yeast, which proliferates by budding, and it was confirmed that the order of microbial inoculation has a significant effect for the growth of both microorganisms (i.e., yeast and Bacillus) in solid fermentation. In particular, it was confirmed that both Bacillus and yeast can more readily utilize the water-soluble saccharide component, because the oligosaccharides in the soybean meal are decomposed during yeast fermentation by first performing yeast fermentation for 6 hours.

Example 6: Problems of Odor Improvement

[0110] A fermented product of Bacillus produces a peculiar odor during the fermentation process due to ammonia, etc., and this may be a limiting factor in using feed. Therefore, in this Example, it was confirmed whether or not the complex fermentation by yeast and Bacillus reduced the odor of the fermented product of Bacillus. An odor test was performed with regard to a mixed raw material of soybean meal and corn gluten, a product fermented by Bacillus amyloliquefaciens (KCCM11471P) alone, and a product of complex fermentation by yeast (bread yeast; CJN1697 or CJN2343) and Bacillus amyloliquefaciens (KCCM11471P) (50 subjects). The score was determined on a point scale of 0 to 5 such that a higher score represents a higher intensity of peculiar odor of Bacillus (Table 6).

[0111] As a result, it was confirmed that the product fermented by Bacillus fermentation alone (use of Bacillus amyloliquefaciens (KCCM11471P)) showed the highest score. Additionally, it was confirmed that when the Bacillus sequential fermentation was performed using CJN1697 or CJN2343 yeast, the odor was significantly reduced compared to when the commercially available yeast was used.

TABLE-US-00006 TABLE 6 Sequential Sequential Sequential Bacillus Fermentation Fermentation Fermentation Fermen- of Yeast and of Yeast and of Yeast and Raw tation Bacillus Bacillus Bacillus Material Alone (Bread Yeast) (CJN1697) (CJN2343) Mean 1.6 4.75 3.8 2.5 2.45 STDEV 0.82 0.55 0.89 1.00 0.76

Example 7: Analysis of Nucleotide Sequence of 18S rRNA Gene and Phylogeny of Saccharomyces cerevisiae Strains of the Present Disclosure

[0112] To analyze the strains isolated in Example 1, 18S ribosomal DNA sequencing was performed in the following manner. The chromosomes of CJN1697 and CJN2343 strains were isolated using the Wizard genomic DNA purification kit (Promega, USA), and then subjected to PCR amplification using NS1 (5-GTAGTCATATGCTTGTCTC-3) and NS8 (5-TCCGCAGGTTCACCTACGGA-3) primers, which are universal primers used in 18S rRNA sequencing. The amplified PCR products were purified using the Wizard SV gel and PCR clean-up system (Promega, USA). As a result, the purified amplified PCR products were compared with the ribosomal DNA sequences of GENEBANK using the BLASTN program, and the sequence homology was compared and analyzed using the Clustal X and Mega 2 programs.

[0113] As a result of the phylogenetic analysis, both strains of the present disclosure (i.e., CJN1697 and CJN2343) showed a 99% homology to that of Saccharomyces cerevisiae, a reference strain (FIGS. 3 and 4). The strains of the present disclosure (i.e., CJN1697 and CJN2343) were each named Saccharomyces cerevisiae CJN1697 and Saccharomyces cerevisiae CJN2343, and deposited to the Korean Culture Center of Microorganisms (KCCM) on Oct. 11, 2017, according to the Budapest Treaty under Accession Numbers KCCM12123P and KCCM12124P, respectively.

[0114] From the foregoing, a skilled person in the art to which the present disclosure pertains will be able to understand that the present disclosure may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present disclosure. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present disclosure. On the contrary, the present disclosure is intended to cover not only the exemplary embodiments but also various alternatives, modifications, equivalents, and other embodiments that may be included within the spirit and scope of the present disclosure as defined by the appended claims.

[Accession Number]

[0115] Name of Depositary Agency: Korean Culture Center of Microorganisms (KCCM)

[0116] Deposit Number: KCCM12123P

[0117] Date of Deposition: Oct. 11, 2017

[0118] Name of Depositary Agency: Korean Culture Center of Microorganisms (KCCM)

[0119] Deposit Number: KCCM12124P

[0120] Date of Deposition: Oct. 11, 2017

[0121] Name of Depositary Agency: Korean Culture Center of Microorganisms (KCCM)

[0122] Deposit Number: KCCM11471P

[0123] Date of Deposition: Oct. 11, 2017