<i>Bacillus </i>sp. strain with improved productivity of fermented soybean meal and method for producing fermented soybean meal using the same

Abstract

The present invention relates to a Bacillus amyloliquefaciens K2G strain, which is excellent in removal of anti-nutritional factors and in protease activity, and shows excellent antimicrobial activity against pathogens and reduced productivity of viscous substances, a method for producing a fermented soybean meal using the strain, a fermented soybean meal produced therefrom, and a feed composition including the same. The fermented soybean meal prepared by Bacillus amyloliquefaciens K2G strain according to the present invention has few anti-nutritional factors such as trypsin inhibitors, soybean oligosaccharides, and polysaccharides, a high content of crude proteins, and high protein solubility, and also consists of small-sized peptides digestible by livestock due to low-molecularization, thereby being effectively used as a high-quality vegetable protein feed having excellent absorption rate and feed efficiency.

Claims

1. A Bacillus amyloliquefaciens strain K2G (Korean Culture Center of Microorganisms Accession No. KCCM11471P) for producing a fermented soybean meal by solid state fermentation.

2. The Bacillus amyloliquefaciens strain K2G according to claim 1, wherein the Bacillus amyloliquefaciens strain K2G has anti-nutritional factors-removing activity, protease activity, antimicrobial activity and reduced production of a viscous substance during fermentation compared to the parent strain (Bacillus amyloliquefaciens), wherein the viscous substance is γ-PGA (Poly-γ-Glutamic acid).

3. A composition comprising the Bacillus amyloliquefaciens strain K2G (Korean Culture Center of Microorganisms Accession No. KCCM11471P) and a suitable carrier for producing a fermented soybean meal by solid state fermentation.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows the results of Example 1 for selecting strains with high protease productivity according to the present invention;

(2) FIG. 2 shows the results of measuring growth inhibition activity of the strains with high protease productivity selected in Example 1 against that of E. coli and Salmonella;

(3) FIG. 3 shows a flow chart for selecting a mutant strain with reduced productivity of viscous substances from the strains with high protease productivity according to the present invention;

(4) FIG. 4 shows the results of concentration-gradient SDS-PAGE for qualitative analysis of γ-PGA content during the selection process of the mutant strain according to the present invention, in which

(5) Lane M: molecular weight marker

(6) Lane 1: 1 g/L γ-PGA standard

(7) Lane 2: 0.5 g/L γ-PGA standard

(8) Lane 3: 0.25 g/L γ-PGA standard

(9) Lane 4: CJ823 strain

(10) Lane 5: U304 mutant strain

(11) Lane 6: U305 mutant strain

(12) Lane 7: U306 mutant strain

(13) Lane 8: U307 mutant strain;

(14) FIG. 5 is a phylogenetic tree showing the phylogenetic relationship of the mutant strain Bacillus amyloliquefaciens K2G selected according to the above procedures; and

(15) FIG. 6 shows the results of SDS-PAGE for hydrolysis degree of fermented soybean meal which is obtained by solid fermentation of Bacillus amyloliquefaciens K2G according to the present invention,

(16) Lane M: molecular weight marker

(17) Lane 1: raw material (raw soybean meal)

(18) Lane 2: TP6 fermentation for 20 hours

(19) Lane 3: K2G fermentation for 16 hours

(20) Lane 4: K2G fermentation for 20 hours.

MODE FOR INVENTION

(21) Hereinafter, the present invention will be described in more detail with reference to Examples. However, it is apparent to those skilled in the art to which the present invention pertains that these Examples are for illustrative purposes only, and the scope of the present invention is not intended to be limited by these Examples.

Example 1: Selection of Strains Having High Protease Productivity

(22) In order to isolate strains having excellent protease productivity, the present inventors isolated approximately 3000 kinds of microorganisms from a variety of traditional fermented foods (kimchi, fermented bean paste, traditional folk wine, salted fish, etc.), and of them, approximately 1300 kinds of feed adaptable (Korea Feed Ingredients Association, probiotics) Bacillus strains were identified. From the strains, it was intended to find multifunctional strains having high protease expression, an antimicrobial activity and a rapid growth rate.

(23) In detail, selection of strains having high protease productivity was performed by comparing the size of clear zone which was formed due to degradation of substrate on 2% (w/v) skim milk (Difco, USA)-containing YM agar plate (yeast extract 3.0 g, malt extract 3.0 g, peptone 10.0 g, agar 20.0 g) (FIG. 1).

(24) The strains having high protease productivity thus selected were inoculated in TSB media (enzymatic digest of casein 17.0 g, enzymatic digest of soybean meal 3.0 g, NaCl 5.0 g, dipotassium phosphate 2.5 g, dextrose 2.5 g, fmal pH: 7.3±0.2 at 25° C.), respectively, followed by culture at 37° C., and 200 rpm for 12 hours. Each 1.0 μl of the culture broths was spotted onto skim milk-containing YM agar plate. The agar plates were incubated at 37° C. for 16 hours and the diameter of the clear zone formed on the plate was measured.

(25) At this time, Bacillus subtilis TP6 (KFCC 11343P) which has been used as a fermentation strain in the conventional method for producing fermented soybean meal by solid fermentation was used as a control group (Korean Patent Publication No. 10-2011-0027535).

(26) TABLE-US-00001 TABLE 1 Name of Number of Diameter of clear Number of Diameter of clear strain strain zone (mm) strain zone (mm) Bacillus TP6 3.30 CJ823 6.23 sp. CJ361 5.46 CJ873 5.48 CJ400 5.88 CJ924 5.69 CJ434 5.34 CJ933 5.20 CJ453 6.40 CJ957 4.98 CJ457 6.10 CJ968 4.87 CJ739 5.86 CJ974 5.00 CJ759 5.43 — —

(27) As shown in Table 1, 14 kinds of strains were selected as strains having high protease productivity, and all of them showed higher protease activity than the Bacillus subtilis TP6.

Example 2: Isolation of Strains Having Inhibitory Activity on Proliferation of Pathogen

(28) In order to isolate strains capable of inhibiting growth or proliferation of E. coli and Salmonella which are the representative pathogens causing food poisoning in livestock and to develop Salmonella-free non-toxic products by applying the strains in the production of fermented soybean meal, 14 kinds of the strains having high protease productivity which were isolated in Example 1 were subjected to measurement of the antimicrobial activity against pathogens.

(29) The antimicrobial activity against pathogens was measured by spot-on-the-lawn test on Salmonella typhymurium (ATCC14028) and E. coli (KCCM11835).

(30) In detail, 14 kinds of the strains having high protease productivity were inoculated in GYP medium (glucose 10.0 g, yeast extract 8.0 g, polypeptone 2.0 g, pH 7.0), respectively, followed by liquid-culture at 37° C. and 180 rpm for 12 hours. Each 1.5 μl of the culture broths of the strains having high protease productivity was spotted onto GYP agar plate (glucose 10.0 g, yeast extract 8.0 g, polypeptone 2.0 g, agar 15.0 g, pH 7.0) to which each 1×10.sup.5 CFU/ml of Salmonella typhymurium ATCC14028 and E. coli ATCC11835 was added, followed by static culture at 37° C. for 15 hours. The size of inhibition zone which was formed around colonies of the strains having high protease productivity spotted onto the plate was examined to determine activity titer. The results are shown in the following Table 2. At this time, Bacillus subtilis TP6 was used as a control group.

(31) TABLE-US-00002 TABLE 2 Name of Name of E. coli Salmonella typhymurium strain strain (KCCM11835) (ATCC14028) Bacillus TP6 + ++ sp. CJ361 ++ ++ CJ400 ++ ++ CJ434 − − CJ453 − − CJ457 − − CJ739 +++ ++ CJ759 − − CJ823 ++++ ++++ CJ873 − − CJ924 +++ ++ CJ933 − − CJ957 − − CJ968 ++ ++ CJ974 +++ +

(32) In Table 2, ‘−’ or ‘+’ is to determine the activity titer, based on the diameter of inhibition zone of live bacteria, and ‘−’ indicates no antimicrobial or antimicrobial activity, ‘+’ indicates that the diameter of inhibition zone is 10.05 mm or less, ‘++’ indicates that the diameter of inhibition zone is 10.05 to 14.05 mm, ‘+++’ indicates that the diameter of inhibition zone is 14.05 to 17.05 mm, and ‘++++’ indicates that the diameter of inhibition zone is 17.05 mm or more.

(33) Antimicrobial spectra of 14 kinds of the strains having high protease productivity against Salmonella and E. coli which are the most common causes of gastrointestinal disease in livestock were examined. As a result, CJ823 showed the most excellent antimicrobial power. The selected CJ823 showed remarkably high antimicrobial activity against both E. coli (++++ vs. +) and Salmonella (++++ vs. ++), compared to the control group Bacillus subtilis TP6.

Example 3: Test for Application of Salmonella Proliferation-Inhibiting Soybean Meal

(34) According to the results of Example 2, CJ823 on the agar plate showed excellent antimicrobial activity against Salmonella. However, the following experiment was performed in order to confirm whether the strain also exhibited the proliferation-inhibiting ability against Salmonella during practical fermentation of the soybean meal.

(35) In detail, both CJ823 and Salmonella typhymurium ATCC14028 were inoculated into soybean meal (moisture content of 45%) which was steamed at 100° C. for 30 minutes, at a density of 4.5×10.sup.7 CFU/g and 1.0×10.sup.3 CFU/g, respectively and changes in the number of cells were examined at 37° C. and constant humidity for 24 hours. At this time, Salmonella typhymurium was singly inoculated into soybean meal, which was used as a control group. The mixture of Salmonella typhymurium and Bacillus subtilis TP6 was inoculated into soybean meal, which was used as a comparison group.

(36) The strains were inoculated, and a predetermined amount of soybean meal was taken before fermentation and after fermentation for 12, 16 and 20 hours. The soybean meal was diluted in 0.8% NaCl sterile solution, and then each 100 μl thereof was applied to an XLD agar plate (yeast extract 3 g, lactose 7.5 g, sucrose 7.5 g, xylose 3.5 g, L-lysine 5 g, ferric ammonium citrate 0.8 g, Phenol Red 0.08 g NaCl 5 g, sodium deoxycholate 2.5 g, sodium thiosulfate 6.8 g, agar 13.5 g, final pH: 7.4±0.2 at 25° C.) to count the number of Salmonella typhymuriutn colony, and the results are shown in the following Table 3.

(37) The ratio between two strains was measured on the assumption that Salmonella typhymurium contamination occurs during proliferation of the main fermentation strain CJ823 in the steamed soybean meal. Actual contamination ratio may differ depending on the environmental conditions for the production of fermented soybean meal.

(38) TABLE-US-00003 TABLE 3 Number of Salmonella typhymurium (×10/g) Salmonella Salmonella Salmonella Fermen- typhymurium typhymurium + typhymurium + tation time alone TP6 CJ823 .sup. 0 hr 2330 2330 2330 12 hrs 13900 2400 2000 16 hrs 1200000 2300 50 20 hrs 3100000 2200 35

(39) As shown in Table 3, in the control group inoculated with only Salmonella typhymurium, as the culture time was increased, the number of the bacteria was steadily increased to 3.1×10.sup.7 CFU/g after 20-hr fermentation. In contrast, in the comparison group inoculated with a mixture with Bacillus subtilis TP6, the number of Salmonella typhymurium was inhibited to 2.2×10.sup.4 CFU/g. In the experimental group inoculated with the mixture with the CJ823 according to the present invention, the number of Salmonella typhymurium was further reduced to 3.5×10.sup.2 CFU/g.

(40) These results indicate that CJ823 according to the present invention effectively inhibits the growth of Salmonella during a practical fermentation process, and thus it is suitable for the production of high-quality fermented soybean meal.

Example 4: Selection of Mutant Strains

(41) CJ823 selected in Examples 1 to 3 produces high concentration of protease and shows excellent antimicrobial activity against pathogens, but produces sticky viscous substances due to polymerization of levan form fructan and polyglutamate which are derived from sugars and proteins of the raw soybean by the enzymes produced during the fermentation process. Excessive production of the viscous substances in a large-scale industrial process hinders agitation, and there are difficulties in the control of dissolved oxygen in the fermented product and temperature, and transfer.

(42) Accordingly, the present inventors caused UV-induced mutations in CJ823 strain as follows, in order to develop a mutant strain having reduced productivity of viscous substances while maintaining or improving its own enzymatic/physiological characteristics. The selection process of the mutant strain is as shown in FIG. 3.

(43) First, CJ823 was plated on TSB agar plate (enzymatic digest of casein 17.0 g, enzymatic digest of soybean meal 3.0 g, NaCl 5.0 g, dipotassium phosphate 2.5 g, dextrose 2.5 g, agar 15.0 g, final pH: 7.3±0.2 at 25° C.) and cultured at 37° C. for 12 hours to activate the strain.

(44) In this culture, 1% of a suspension of the seed microorganism was inoculated into TSB media (enzymatic digest of casein 17.0 g, enzymatic digest of soybean meal 3.0 g, NaCl 5.0 g, dipotassium phosphate 2.5 g, dextrose 2.5 g, final pH: 7.3±0.2 at 25° C.) prepared in advance, and cultured with shaking at 37° C. and 180 rpm. After culture, the culture broth was centrifuged at 25° C. and 8000 rpm for 10 minutes to separate the cell pellet and supernatant. Only the cell pellet was taken and washed with 0.8% NaCl sterile solution. After washing, artificial mutation was induced by UV irradiation (254 nm) to the recovered cell pellet using a UV lamp (VIBER LOURMAT, 115 V, 60 Hz).

(45) The cell was plated on a 2% skim milk-containing TSA agar plate (enzymatic digest of casein 15 g, enzymatic digest of soybean meal 5 g, NaCl 5 g, agar 15 g, final pH 7.3±0.2 at 25° C.) which is a selection medium, and cultured at 37° C. for 20 hours. After culture, the size (diameter, mm) of clear zone formed was determined using Caliper (CD-20CPX, Mitutoyo, Kanagawa, Japan), and 16 kinds of mutant strains having a high proteolytic activity were primarily selected.

(46) Subsequently, each of the 16 kinds of mutant strains thus selected was inoculated into heat-treated soybean meal, and cultured for 20 hours, and the culture broth obtained therefrom was centrifuged at 25° C. and 8000 rpm for 10 minutes to separate the cell pellet and supernatant. The γ-PGA content in the supernatant was determined by measuring the weight of γ-PGA which was recovered after isolation and purification and freeze-drying according to the method disclosed in the document (Goto et al. Biosci. Biotechnol. Biochem, 56:1031-1035, 1992).

(47) The protease activity was measured according to the method described in Example 1, and the γ-PGA content was measured by the following two methods of qualitative and quantitative analysis.

(48) First, for qualitative analysis of γ-PGA activity, 40 μl of the separated supernatant was mixed with 10 μl of 5× staining buffer solution, and then loaded on a 5 to 20% gradient SDS-polyamide gel to perform concentration gradient SDS-PAGE. After electrophoresis, the standard protein was stained with a Coomassie dye reagent, followed by destaining. Then, poly-γ-glutamic acid was stained with methylene blue to perform qualitative analysis.

(49) As shown in FIG. 4, poly-γ-glutamic acid was detected in the supernatant of CJ823 (parent strain) of Lane 4, but the polymer-producing ability was greatly reduced and high protease activity was observed in the supernatant of U304 mutant strain of Lane 5.

(50) Meanwhile, for quantitative analysis of γ-PGA content, the supernatant of solid fermentation was diluted with an equal amount of distilled water, and then centrifuged at 20,000×g for 20 minutes. pH of the obtained supernatant was adjusted to 3.0 using 6 M HCl, and left at 4° C. for one day. Thereafter, the supernatant was centrifuged at 25,000×g for 30 minutes, and then the pellet was recovered. The pellet was completely dissolved in 100˜200 times volume of distilled water, and centrifuged at 25,000×g for 30 minutes to remove impurities. Salts were removed by dialysis at 4° C. for one day. The resultant was freeze-dried to recover γ-PGA, and the results obtained therefrom are shown in the following Table 4.

(51) TABLE-US-00004 TABLE 4 Mutant strain γ-PGA (g/L) Protease (U/g) TP6 1.61 150 CJ823 25.36 450 U101 11.19 330 U102 13.36 220 U103 14.83 320 U304 4.96 500 U305 24.50 400 U306 16.70 280 U307 5.52 120 U609 6.13 15 U610 11.09 450 U611 21.77 260 U612 12.05 400 U813 10.12 180 U814 23.13 370 U815 1.06 260 U816 5.26 100 U1019 13.01 400

(52) As shown in Table 4, most of the mutant strains showed low γ-PGA content, compared to CJ823. Of them, the U304 mutant strain showing the highest protease activity and relatively low γ-PGA content was finally selected. In particular, the U304 mutant strain showed lower γ-PGA content and three times or more higher protease activity than the control group, Bacillus subtilis TP6.

(53) The fmally selected U304 mutant strain is characterized in that it has reduced productivity of viscous substances by remarkably reducing production of polymers such as poly-γ-glutamic acid, while maintaining the characteristics of the parent strain, including production of high concentration of protease and excellent antimicrobial activity against pathogens.

Example 5: Determination of 16S rRNA Sequence of U304 Mutant Strain (K2G) and Phylogenetic Analysis

(54) In order to identify the U304 mutant strain having reduced productivity of viscous substances, the strain was inoculated into a new NA agar plate and cultured at 37° C. for 16 hours. The colonies formed were diluted with 0.8% NaCl sterile solution, and then injected into a BCL ID card (bioMNitek Inc., Hazewood, USA) consisting of 63 kinds of dry media and biochemical reactants, and the results were integrally stored in VITEK 2 Compact software (bioMVitek) every 15 minutes, and identification was completed after 14 hours.

(55) As a universal primer for bacterial identification by 16S rRNA sequence analysis, 518F (5′-CCAGCAGCCGCGGTAATACG-3′) and 800R (5′-TACCAGGGTATCTAATCC-3′) of SEQ ID NOs. 2 and 3 were used, and after amplification of 16S rRNA by PCR, 1,333 bp containing a base sequence of 50900 bp which is important in identification was translated using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems Inc., USA). The result of 16S rRNA sequence analysis showed that the U304 mutant strain has 16S rRNA sequence of SEQ ID NO. 1.

(56) Sequence similarity was determined by Blast similarity search program (National Institute of Biotechnology Information) and the position was determined in a phylogenetic tree after multiple sequence alignment (FIG. 5).

(57) TABLE-US-00005 TABLE 5 Re- Re- Characteristics sult Characteristics sult β-Xylosidase + D-Mannitol + L-Lysine-arylamidase − D-Mannose + L-Aspartate arylamidase − D-Melezitose − Leucine arylamidase (+) N-Acetyl-D-glucosamine − Phenylalanine arylamidase + Palatinose (+) L-Proline arylamidase − L-Rhamnose − β-Galactosidase − β-Glucosidase (+) L-Pyrrolydonyl- arylamidase + β-Mannosidase − α-Galactosidase + Phosphoryl choline − Alanine arylamidase + Pyruvate + Tyrosine arylamidase + α-Glucosidase − β-N-Acetyl-glucosaminidase (−) D-Tagatose − Ala-Phe-Pro arylamidase + D-Trehalose (+) Cyclodextrine − Insulin − D-Galactose − D-Glucose (+) Glycogene − D-Ribose − myo-Inositol − Putrescine assimilation − Methyl-A-D-glucopyranoside + Kanamycin resistance (−) acidification Ellman − Oleandomycin resistance − Methyl-D-xylosdie − Esculin hydrolyse + α-Mannosidase − Tetrazolium Red − MALTOTRIOSE − Plomixin_B resistance − Glycine ARYLAMIDASE (−)

(58) The results of 63 kinds of biochemical tests were analyzed by Vitec 2 Compact Software. As a result, the strain was classified as Bacillus subtilis/Bacillus amyloliquefaciens with a probability of 98%. As shown in FIG. 5, the results of phylogenetic analysis showed that U304 mutant strain has the closest relationship with the standard strain, Bacillus amyloliquefaciens subsp. plantarum FZB42T (CP000560) and 16S rDNA sequence homology was 99.92% (1332 bp/1333 bp).

(59) Therefore, when the biochemical characteristics and the results of the phylogenetic analysis were taken together, the U304 mutant strain according to the present invention was designated as Bacillus amyloliquefaciens K2G, and deposited under Budapest Treaty to the Korea Culture Center of Microorganisms (KCCM) on Nov. 7, 2013 with Accession No. KCCM11471P.

Example 6: Changes in Crude Protein Content According to Fermentation Time Upon Fermentation of Soybean Meal Using Bacillus amyloliquefaciens K2G

(60) In order to examine changes in the crude protein content according to fermentation time upon fermentation of soybean meal using Bacillus amyloliquefaciens K2G according to the present invention, the following experiment was performed.

(61) First, 400 g of soybean meal was prepared to have the water content of 45%, and steamed at 100° C. for 30 minutes, and then cooled to 40° C. or lower. Subsequently, the Bacillus amyloliquefaciens K2G was plated on TSB agar plate (enzymatic digest of casein 17.0 g, enzymatic digest of soybean meal 3.0 g, NaCl 5.0 g, dipotassium phosphate 2.5 g, dextrose 2.5 g, agar 15.0 g, final pH: 7.3±0.2 at 25° C.), and cultured at 37° C. for 12 hours to activate the strain. Approximately 2 loops of the activated strain was suspended in 9 ml of 0.8% NaCl sterile solution (diluted to 0.2 at A.sub.660nm), and this suspension was used as a seed microorganism. In this culture, 1% of the suspension of the seed microorganism was inoculated into 40 ml of TSB media (enzymatic digest of casein 17.0 g, enzymatic digest of soybean meal 3.0 g, NaCl 5.0 g, dipotassium phosphate 2.5 g, dextrose 2.5 g, final pH: 7.3±0.2 at 25° C.) prepared in advance, and cultured with shaking at 37° C. and 180 rpm.

(62) 40 ml of the culture broth of the Bacillus amyloliquefaciens K2G was prepared to have the water content of 45%, added to the steamed soybean meal, and mixed well, followed by static culture at 37° C. and constant humidity for 20 hours. The culture sample was dried in a 60° C. dryer until the water content reached 10% or less, and then the crude protein content in each sample was measured using a Kjeldahl system (Kjltec 2100). At this time, the crude protein content was measured before fermentation and after fermentation for 12, 16 and 20 hours, and Bacillus subtilis TP6 was used as a control group. The crude protein content (%, correction to 10% moisture) measured therefrom is shown in the following Table 6. At this time, Bacillus subtilis TP6 was used as a control group.

(63) TABLE-US-00006 TABLE 6 TP6 (%, correction K2G (%, correction Section to 10% moisture) to 10% moisture) Raw soybean meal 50.86 ± 0.15 50.86 ± 0.15 Steamed soybean meal 50.91 ± 0.12 50.91 ± 0.12  0-hr fermentation 51.19 ± 0.38 51.19 ± 0.38 12-hr fermentation 54.21 ± 1.18 55.59 ± 0.66 16-hr fermentation 55.07 ± 0.79 56.46 ± 0.19 20-hr fermentation 55.86 ± 0.75 57.32 ± 0.28 24-hr fermentation 56.36 ± 0.80 58.02 ± 0.45

(64) As shown in Table 6, based on 24-hr fermentation, the soybean meal fermented by Bacillus amyloliquefaciens K2G according to the present invention showed the crude protein content equivalent to or higher than the soybean meal fermented by Bacillus subtilis TP6 which is known as the conventional soybean meal fermentation strain.

(65) These results indicate that Bacillus amyloliquefaciens K2G according to the present invention can be effectively used in the production of fermented soybean meal as a high-quality protein feed.

Example 7: Changes in TI Content According to Fermentation Time Upon Fermentation of Soybean Meal Using Bacillus amyloliquefaciens K2G

(66) In order to examine changes in the trypsin inhibitor (TI) content according to fermentation time upon fermentation of soybean meal using Bacillus amyloliquefaciens K2G according to the present invention, the following experiment was performed.

(67) Specifically, as in Example 6, 400 g of soybean meal was prepared to have the water content of 45%, and steamed at 100° C. for 30 minutes, and then Bacillus amyloliquefaciens K2G was inoculated into the steamed soybean meal at a density of 4.5×10.sup.7 CFU/ml, followed by culture at 37° C. and constant humidity for 24 hours. The TI content was measured according to AACC-71-10 (American association of cereal chemists, 1995) before fermentation and after fermentation for 16, 20 and 24 hours, and the TI content (mg/g) measured therefrom is shown in the following Table 7. At this time, Bacillus subtilis TP6 was used as a control group.

(68) TABLE-US-00007 TABLE 7 Section TP6 (TI, mg/g) K2G (TI, mg/g) Raw soybean meal 4~5.sup.  4~5.sup.  Steamed soybean meal 2~2.5 2~2.5 16-hr fermentation 1.26 0.39 20-hr fermentation 0.53 0.00 24-hr fermentation 0.38 0.00

(69) As shown in Table 7, the soybean meal fermented by Bacillus amyloliquefaciens K2G according to the present invention showed TI content of 0.39 mg/g after 16-hr fermentation, which corresponds to that in the soybean meal fermented for 24 hours by Bacillus subtilis TP6 which is known as the conventional soybean meal fermentation strain, suggesting that the reduction in anti-nutritional factors can be achieved at an equivalent level by only 16-hr fermentation.

(70) The present invention is excellent in that the reduction in fermentation time increases the turnover rate of the fermentor to increase the number of batches annually producible, and consequently, consumers can be provided with high-quality fermented soybean meals at a lower cost.

Example 8: Measurement of Hydrolysis Degree of Soybean Protein and its Solubility in KOH Upon Fermentation of Soybean Meal Using Bacillus amyloliquefaciens K2G

(71) Soybean meal is a vegetable feed raw material having high content of proteins, but has a disadvantage that it is insufficient for, in particular, young livestock, due to anti-nutritional factors and proteins having a low digestion and absorption rate. One of methods for improving this disadvantage is to prepare the proteins in the form of hydrolyzed peptides or to degrade it into low-molecular weight proteins being easily digestible by fermentation using a microorganism. Bacillus amyloliquefaciens K2G according to the present invention is a strain having high protease productivity, and thus it was expected that soybean proteins in the fermented soybean meal prepared by using the same can be degraded by protease secreted from the strain.

(72) In order to confirm this, the hydrolysis degree of the fermented soybean meal according to the present invention was first measured. As in Example 6, 400 g of soybean meal was prepared to have the water content of 45%, and steamed at 100° C. for 30 minutes, and then Bacillus amyloliquefaciens K2G was inoculated into the steamed soybean meal at a density of 4.5×10.sup.7 CFU/ml, followed by culture at 37° C. and constant humidity for 24 hours. The culture broth obtained therefrom was centrifuged at 25° C. and 8000 rpm for 10 minutes to separate the cell pellet and supernatant. 40 μl of the separated supernatant was mixed with 10 μl of 5× staining buffer solution, and then SDS-PAGE (10%) was performed to examine migration of the proteins according to their molecular weight. After electrophoresis, the polyamide gel was stained with Coomassie Brilliant R250 to examine the composition and molecular weight of the protein, and the results are shown in FIG. 6. At this time, raw soybean meal and fermented soybean meal prepared by Bacillus subtilis TP6 were used as control groups.

(73) As shown in FIG. 6, it was found that the fermented soybean meal prepared by Bacillus amyloliquefaciens K2G according to the present invention showed a high protein density in the low molecular weight region, compared to the raw soybean meal and the fermented soybean meal prepared by another strain. On SDS-polyamide gel, upper bands indicate high-molecular weight proteins and lower bands indicate low-molecular weight proteins. These results indicate that in the case of soybean meals having the same molecular weights, high-molecular weight proteins in the soybean meal fermented by Bacillus amyloliquefaciens K2G according to the present invention are hydrolyzed into low-molecular weight proteins.

(74) According to the previous reports, dietary supplementation of broiler chickens was performed four times using the soybean meal showing KOH (potassium hydroxide) solubility of 80% and the soybean meal showing lower solubility (55% to 68%). As a result, the weight, feed intake, and feed efficiency of broiler chickens which were fed with the soybean meal showing low KOH solubility were remarkably low or decreased, compared to the broiler chickens fed with the soybean meal showing KOH solubility of 80% (Abulto et al. J. Appl. Poult. Res. 7:189-195, 1998b).

(75) Therefore, KOH solubility of the fermented soybean meal according to the present invention was measured according to the method disclosed in the document (Parsons et al. J Anim Sci., 69: 2918-24, 1991). Briefly, 1.0 g of the fermented soybean meal thus prepared was added to 0.2% KOH solution and mixed for 20 minutes, followed by filtration. The nitrogen content in the filtrate was measured using a Kjeldahl system and converted to solubility. The results are shown in Table 8.

(76) TABLE-US-00008 TABLE 8 Section TP6 (KOH, %) K2G (KOH, %) Raw soybean meal 80.17 ± 2.38 80.17 ± 2.38 Steamed soybean meal 73.35 ± 3.32 73.35 ± 3.32 16 hr-fermentation 78.38 ± 2.85 85.03 ± 3.07 24 hr-fermentation 78.99 ± 3.01 85.20 ± 3.60

(77) As shown in Table 8, KOH solubility of the steamed soybean meal was reduced from 80% to 70%, but KOH solubility was increased to 85% during solid fermentation by Bacillus amyloliquefaciens K2G according to the present invention. These results suggest that KOH was increased due to degradation of soybean proteins by protease secreted from Bacillus amyloliquefaciens K2G according to the present invention.

INDUSTRIAL APPLICABILITY

(78) The Bacillus amyloliquefaciens K2G strain (KCCM11471P) according to the present invention is excellent in removal activity of anti-nutritional factors, in protease activity, and in antimicrobial activity against pathogens, and has reduced productivity of viscous substances during fermentation. Thus, when the strain is used as a seed microorganism to perform solid fermentation of soybean meal, high-quality fermented soybean meal with improved digestion and absorption rates and feed efficiency can be produced due to low-molecularization by hydrolysis of soybean proteins and an increase in the content of crude proteins, inactivation of trypsin inhibitors, or a reduction in the content of anti-nutritional factors such as non-digestible polysaccharides.