FERMENTED SOYBEAN MEAL AND PREPARATION METHOD THEREFOR

Abstract

The present invention relates to a method for preparing fermented soybean meal, and more specifically, relates to a method for preparing fermented soybean meal using a facultative anaerobic soybean meal fermenting microorganism, fermented soybean meal prepared therefrom, and a feed composition comprising the same. In addition, the present invention relates to a novel Enterococcus sp. strain, and more specifically, relates to an Enterococcus faecium strain.

Claims

1. A method for preparation of fermented soybean meal, comprising a step of obtaining soybean meal extract solution and residual soybean meal by extracting raw soybean meal with an extraction solvent, and a step of performing solid culture of a fermentation raw material comprising one or more kinds selected from the group consisting of raw soybean meal and the residual soybean meal, by using a microorganism fermenting soybean meal.

2. The method for preparation according to claim 1, wherein the microorganism fermenting soybean meal is a facultative anaerobic lactic acid bacterium, and the step of solid culture does not comprise an oxygen aeration process.

3. (canceled)

4. The method for preparation according to claim 1, wherein the extraction solvent is one or more kinds selected from the group consisting of water and alcohols of 1 to 6 carbon atoms, and is used at a weight ratio of 1 to 10 times of the raw soybean meal.

5. The method for preparation according to claim 1, wherein the extraction solvent is 20 to 70° C. of temperature and pH 2 to 8.

6. The method for preparation according to claim 1, wherein the residual soybean meal having a water content of 80 w/w % or less is obtained by extracting the raw soybean meal with an extraction solvent and then separating by a centrifugation process.

7. The method for preparation according to claim 1, wherein the fermentation raw material is a mixture of the residual soybean meal and raw soybean meal, and the crude protein content or anti-nutritional factor content of the fermented soybean meal is controlled by adjusting a mixing ratio.

8. The method for preparation according to claim 7, wherein the anti-nutritional factor is one or more kinds selected from the group consisting of trypsin inhibitor, beta-conglycininin, indigestible oligosaccharide, hemagglutinin (lectin), saponin and tannin.

9. The method for preparation according to claim 1, wherein the fermentation raw material is a mixture in which the raw soybean meal and residual soybean meal are mixed at a weight ratio of 1:10 to 10:1.

10. The method for preparation according to claim 1, wherein the fermentation raw material is obtained from soybean meal, and includes 20 to 48% (w/w) of the crude protein content, and 0.6 to 1.7(w/w %) of the indigestible oligosaccharide content, and wherein the fermented soybean meal has the crude protein content of higher than 46% (w/w) to lower than 80% (w/w).

11. The method for preparation according to claim 1, comprising a step of obtaining soybean meal extract solution and residual soybean meal obtained by extracting raw soybean meal with an extraction solvent; a step of preparing a fermentation raw material in which the crude protein content is 20 to 48% (w/w) and the indigestible oligosaccharide content is 0.6 to 1.7(w/w %), by mixing the raw soybean meal and residual soybean meal, and a step of performing solid culture of the fermentation raw material, by using a microorganism fermenting soybean meal.

12. The method for preparation according to claim 1, wherein the microorganism fermenting soybean meal is one or more kinds selected from the group consisting of Enterococcus sp. strain, Weissella sp. strain, and Lactobacillus sp. strain.

13. (canceled)

14. (canceled)

15. A soybean meal fermented product, which is obtained by fermenting a fermentation raw material comprising one or more kinds selected from the group consisting of raw soybean meal and residual soybean meal by using a facultative anaerobic soybean meal fermenting microorganism, wherein the fermentation raw material comprises 20 to 48% (w/w) of the crude protein content and 0.6 to 1.7 (w/w %) of the indigestible oligosaccharide content, and wherein the soybean meal fermented product comprises 0.0001 to 8 (mg/g) of trypsin inhibitor, 0 to 70,000 (ppm) of beta-conglycinin, 0.0001 to 1.7 (w/w %) of indigestible oligosaccharide, or 46 to 80% (w/w) of crude protein.

16. The soybean meal fermented product according to claim 15, wherein the fermentation raw material comprises one or more kinds selected from the group consisting of raw soybean meal and residual soybean meal which is a solid component obtained by removing soybean meal extract solution in a solvent extract of soybean meal.

17. (canceled)

18. An animal feed composition comprising the fermented product of soybean meal according to claim 15.

19. The feed composition according to claim 18, wherein the animal is one or more kinds selected from the group consisting of pig, cow, chicken, duck, goat, sheep, dog and cat.

20. The feed composition according to claim 18, wherein (1) the fermented product of soybean meal has the crude protein content of 48 to 53% (w/w), and the animal is adult, (2) the fermented product of soybean meal has the crude protein content of 50 to 60% (w/w), and the animal is a piglet or chick, or (3) the fermented product of soybean meal has the crude protein content of 53 to 65% (w/w), and the animal is fish.

21. (canceled)

22. (canceled)

23. The method for preparation according to claim 1, wherein in the fermented soybean meal, the content of protein having a molecular weight of lower than 25 kD is 25 to 99.9% by weight, and the content of protein having a molecular weight of 25 to lower than 50 kD is 0.01 to 60% by weight, and the content of protein having a molecular weight of 50 kD or more is 0.01 to 30% by weight, based on 100% by weight of the total protein in the fermented soybean meal.

24. An Enterococcus faecium strain having the optimum growth temperature of 40 to 45° C.

25. (canceled)

26. The strain according to claim 24, wherein the strain is Enterococcus faecium SLB130 strain deposited with accession number of KCTC13566BP.

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)

35. (canceled)

36. The method for preparation according to claim 1, further comprising a step of seed culturing that cultures the strain in the soybean meal extract solution obtained by extracting the raw soybean meal with an extraction solvent, before performing the step of fermenting.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0161] FIG. 1 is the result of analyzing the protein in the soybean meal before fermentation and soybean meal after fermentation by SDS-PAGE, and A of FIG. 1 is the result of SDS-PAGE analysis of the protein sample of the raw soybean meal before fermentation, and B of FIG. 1 is the result of SDS-PAGE analysis of the protein sample of the fermented soybean meal in which the raw soybean meal is fermented by using SLB120 strain (Example 4-2), and C to E of FIG. 1 are the results of SDS-PAGE of the protein sample of the fermented soybean meal in which the residual soybean meal is fermented by using SLB130 strain (Example 5-1), and F of FIG. 1 is a size marker and each line represents the sizes of 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kD in order from the top, and G of FIG. 1 is the result of SDS-PAGE analysis of the protein sample of the fermented soybean meal in which the raw soybean meal is fermented by using SLB130 strain (Example 4-1).

MODE FOR INVENTION

[0162] Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are intended to illustrate the present invention only, but the scope of the present invention is not limited by these examples.

EXAMPLE 1

Optimization of Soybean Meal Extraction Conditions

1-1: Weight Ratio of Raw Soybean Meal and Extraction Solvent

[0163] Water at a room temperature was used as an extraction solvent of soybean meal. Soybean meal extraction solution was prepared by varying the weight ratio of raw soybean meal and the extraction solvent to 2.5, 3, 4, 5, 7, 10 and 20 of the extraction solvent weight based on 1 of the soybean meal weight.

[0164] Specifically, water of pH 7.0 and temperature 40° C. was added to soybean meal 100 g (moisture 12%, crude protein content 46%) and it was extracted for 30 min and filtered with a 50 mesh sieve to obtain filtrate which was used as soybean meal extract solution.

[0165] The sugar concentration (Brix) of the soybean meal extract solution was measured by using a saccharometer. In addition, the protein content of the extract solution obtained after extraction was quantified by Bradford method. The crude protein content of residual protein was calculated by excluding the crude protein content extracted by extraction (protein content in the extract solution) from the crude protein content present in the raw soybean meal.

[0166] The result of analysis of the sugar content of the soybean meal extract solution and the protein content of the residual soybean meal was shown in the following Table 1. In Table 1, 1 Bris was defined as the g number of sugars comprised in 100 g of solution and the amount of protein was expressed as mass (g) of protein contained per 100 g of extract solution.

TABLE-US-00001 TABLE 1 Sugar con- Mass ratio centration Total sugars Total protein Protein of of soybean of extract in extract in extract residual meal:extrac- solution solution solution soybean meal tion solvent (Brix) (g) (g) (w/w %)   1:2.5 12.0 15.6 1.2 53.8 1:3 10.0 18.0 1.3 55.4 1:4 7.0 19.6 1.4 56.5 1:5 4.5 17.6 1.4 55.1 1:7 3.0 17.9 1.5 55.2  1:10 2.2 19.14 1.6 56.0  1:20 1.2 22.46 1.7 58.4

[0167] As a result, it was confirmed that when the soybean meal and extraction solvent were extracted at a weight ratio of 1:4, the optimum conditions of the soybean meal extract solution were achieved, which were 7 Brix of the sugar concentration of the soybean meal extract solution, 19.6 g of the total sugars of the entire extract solution, and 1.4% (w/w) of the total protein concentration in the extract solution. When a larger amount of solvent was used, the total amount of indigestible sugars extracted from the raw soybean meal was increased, but the amount of aqueous protein eluted together was also increased, and this reduced the crude protein content of the final fermented soybean meal and also reduced the output of fermented soybean meal. In addition, in consideration of a disadvantages in the process, when a large amount of extraction solvent was used, it could be seen that the weight ratio of about 1:4 was preferable.

[0168] 1-2: Establishment of pH Condition of Extraction Solvent

[0169] To establish the optimum extraction condition for preparation of the soybean meal extract, after setting pH and temperature conditions of the extraction solvent variously, components of the soybean meal extract solution obtained by extracting soybean meal were analyzed. The result was shown in Table 2. The extraction time was 30 min, and in case that the temperature of the extraction solvent was 60° C. or 80° C., when the extraction time was 30 min, the extraction was not good, and thus it was extracted for 15 min.

TABLE-US-00002 TABLE 2 Sugar Sugar Protein Extraction Soybean concentration content of Protein of residual solvent Extraction meal:extraction of extract extraction of extract soybean temperature time solvent ratio solution solution solution meal pH (° C.) (min) (w/w) (Brix) (g) (g) (w/w %) 3 40 30 1:4 6.7 18.8 1.2 56.0 4 40 30 1:4 6.9 19.3 1.3 56.3 5 40 30 1:4 6.8 19.0 1.4 56.1 7 40 30 1:4 6.9 19.3 1.5 56.2 9 40 30 1:4 6.7 18.8 2.2 55.4 10 40 30 1:4 6.8 19.0 3.8 54.7 7.0 40 30 1:4 6.9 19.3 1.5 56.2 7.0 60 15 1:4 6.9 19.3 1.5 56.2 7.0 80 15 1:4 7.1 19.9 1.8 56.4

[0170] As the result of analysis of components, it could be seen that it was preferable that the pH of the extraction solvent was 8 or less, since problems that the crude protein content of the final fermented soybean meal was lowered and the output was reduced finally, as the elution of protein of soybean meal was increased as the pH of the extraction solvent was increased and thereby the crude protein content of the residual soybean meal was lowered.

EXAMPLE 2

Crude Protein Content of Soybean Meal Extract Solution According to pH and Temperature Conditions of Extraction Solvent

[0171] By measuring the crude protein content of the soybean meal extract solution according to pH and temperature conditions of the extraction solvent, the optimum conditions of the extraction solvent for controlling the crude protein content of the residual soybean meal were established.

[0172] Specifically, water was used as the extraction solvent, and hydrochloric acid was added as a pH regulator for adjusting the pH of the extraction solvent. The weight ratio of the raw soybean meal and extraction solvent was 1:4, and after extracting raw soybean meal using extraction solvents of the pH and temperature conditions of Table 3, the crude protein content of the soybean meal extract solution was measured.

TABLE-US-00003 TABLE 3 Sugar Sugar Protein Soybean concentration content of Protein of residual meal:extraction of extract extract of extract soybean solvent ratio Temperature solution solution solution meal Classification (w/w) pH (° C.) (Brix) (g) (g) (w/w %) Example2-1 1:4 3.5 27 6.8 19.0 1.4 56.1 Example2-2 1:4 7.8 27 6.8 19.0 1.5 56.0 Example2-3 1:4 7.8 60 6.9 19.3 1.6 56.1 Example2-4 1:4 7.8 80 6.9 19.3 1.9 56.0

[0173] As a result, it could be seen that it was preferable that the pH of the extraction solvent was 4 or less, as the crude protein content was increased, when pH was high.

[0174] In addition, it could be seen that it was preferable as the temperature of the extraction solvent was low, since the extracted crude protein content was increased as the temperature of the extraction solvent was increased. In particular, it was confirmed that even when the temperature of the extraction solvent was set to 25 to less than 40° C. in case that pH was 4 or less, anti-nutritional factors such as indigestible oligosaccharides, etc. were removed sufficiently at the equal level to the high temperature extraction. In other words, it was confirmed that the efficient removal of anti-nutritional factors of raw soybean meal was possible, since in case that pH was 4 or less, even when the temperature of the extraction solvent was relatively low, there was few effects on the sugar content extracted from the raw soybean meal and the protein content extracted from the raw soybean meal was small.

EXAMPLE 3

Separation and Identification of Soybean Meal Fermenting Microorganism

3-1: Preparation of Soybean Meal Extract Solution

[0175] To establish a soybean meal fermenting microorganism of which growth and development were excellent in the soybean meal extract solution obtained by extracting raw soybean meal with an extraction solvent, soybean meal extract solution was prepared and used as a medium.

[0176] Specifically, under the optimum extraction conditions established in Example 1, the soybean meal extract solution was prepared as follows. Water of 25° C. of which pH was adjusted to 3.5 by adding hydrochloric acid was used as the extraction solvent.

[0177] The extraction solvent was added to the soybean meal and then it was stirred enough, and in 10 min, it was filtered with a 50 mesh sieve to obtain the soybean meal extract solution.

[0178] 3-2: Separation of Soybean Meal Fermenting Microorganism using Soybean Meal Extract Solution

[0179] To separate a microorganism having the ability of fermenting soybean meal, the followings were carried out.

[0180] A candidate strain having the ability of fermenting soybean meal was extracted by using an MRS lactic acid bacteria selective medium, from the soybean meal which water was added in and was left outside for a week or more.

[0181] That the soybean meal extract solution prepared in Example 3-1 was filtered by using a sterilized membrane filter of pore size 0.22 um was used as a medium, and the candidate strain was liquid cultured overnight in the MRS medium, and as a control group, a Bacillus strain was added to each medium at the concentration of 1%, which was liquid cultured overnight in Nutrient broth medium. Each microorganism was grown and developed at the temperature of 40° C. and 45° C. and cultured for 12 hours, and then the viable cell count was measured.

[0182] 3-3: Identification of Soybean Meal Fermenting Microorganism

[0183] According to the result of measurement of the viable cell count, the microorganism showing the high growth and development speed was selected, and 16s rRNA of the selected microorganism was amplified with the primer pair having SEQ ID NOs: 2 and 3 of the following Table 4, and through sequencing, the microorganism identification was conducted. The result was same as 16s rRNA, the sequence of Table 4 below, and the identification of the strain was finally completed by using this sequence information and multiply comparing with BLAST program.

TABLE-US-00004 TABLE 4 Name Sequence listing (5′.fwdarw.3′) Entero- ACGCGGGCGGCGTGCCTAATACATGCAAGTCGTACGCT coccus TCTTTTTCCACCGGAGCTTGCTCCACCGGAAAAAGAGG faecium AGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCC SLB130 ATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATAC (SEQ ID CGTATAACAATCGAAACCGCATGGTTTTGATTTGAAAG NO: 1) GCGCTTTCGGGTGTCGCTGATGGATGGACCCGCGGTGC ATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCAC GATGCATAGCCGACCTGAGAGGGTGATCGGCCACATTG GGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGC AGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGA GCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAA AACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAACT GTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGC TAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGC AAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCA GGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCA ACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGC AGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAAT GCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGG CTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGT GGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGC CGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCC CTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTG GGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTG ACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAAT TCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATC CTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGG CAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTG TCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCTTATTGTTAGTTGCCATCATTCAGTTGGGCACTCTA GCAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGAT GACGTCAAATCATCATGCCCCTTATGACCTGGGCTACA CACGTGCTACAATGGGAAGTACAACGAGTTGCGAAGTC GCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTC GGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAAT CGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACG TTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAG AGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTG GAGCCAGCCGCCTAAGGTGGGATAGATGATGGGGGTGA AGTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGG ATCCCC universal GGTTAGATACCCTGGTA forward primer 785F (SEQ ID NO: 2) universal CCGTCAATTCMTTTRAGTTT reverse primer 785F (SEQ ID NO: 3)

[0184] The selected Enterococcus faecium was named Enterococcus faecium SLB130, and was deposited to the international depository institution under Budapest Treaty, Korean Collection for Type Culture on June 29, 2018 and the accession number, KCTC13566BP was given.

[0185] 3-4: Condition of Growth and Development of Soybean Meal Fermenting Microorganism

[0186] The result of growth and development of Enterococcus faecium SLB130 strain in the soybean meal extract solution was shown in Table 5 and Table 6, and as a control group, Enterococcus faecium SLB120 (accession number: KCTC12868BP) and Bacillus megaterium were used. The OD value and viable cell count of the culture solution and the pH of the culture solution measured after culturing the strain for 12 hours were shown in Table 5 and Table 6. Table 5 showed the result of growth and development in the 40° C. soybean meal extract solution, and Table 6 showed the result of growth and development in the 45° C. soybean meal extract solution.

TABLE-US-00005 TABLE 5 Viable cell count Strain name OD600 pH (cfu/ml) Enterococcus faecium SLB130 3.42 4.8 6.3 × 10.sup.9 Enterococcus faecium SLB120 2.65 5.5 1.4 × 10.sup.8 Bacillus megaterium 0.32 6.5 2.1 × 10.sup.6

TABLE-US-00006 TABLE 6 Viable cell count Strain name OD600 pH (cfu/ml) Enterococcus faecium SLB130 3.31 4.9 6.2 × 10.sup.9 Enterococcus faecium SLB120 1.73 6.1 4.2 × 10.sup.7 Bacillus megaterium 0.19 6.5 1.2 × 10.sup.6

[0187] As a result, as shown in Table 5 and Table 6, the selected strain showed excellent growth and development compared to the control group at the temperature of 40° C. and 45° C., compared to the conventionally separated Enterococcus faecium SLB120 which was used as the control group and bacillus subtilis Bacillus megaterium known to secret a great deal of organic matter decomposing enzymes. The optimum temperature of growth and development of the selected strain SLB130 was shown as 40 to 45° C.

EXAMPLE 4

Preparation of Fermented Soybean Meal using Raw Soybean Meal

Example 4-1 Preparation of Fermented Soybean Meal using Raw Soybean Meal (1)

[0188] Using the soybean meal fermenting microorganism selected in Example 3, the result of fermentation under various soybean meal conditions was measured.

[0189] Specifically, using the raw soybean meal before extraction used in Example 1 as a fermentation raw material, the soybean meal fermenting microorganism selected in Example 3, Enterococcus faecium SLB130 (KCTC13566BP) was inoculated to prepare fermented soybean meal. Specifically, the fermentation was conducted by inoculating Enterococcus faecium SLB120 at the concentration of 2.1×10.sup.8 to the fermentation raw material in an open type tray solid fermenter and starting the fermentation at the temperature of 30° C., thereby increasing the temperature by 40° C., and carrying out the fermentation for 24 hours. During the fermentation process, oxygen was not supplied. Then, it was dried in a tray drier of 50° C. temperature, and the components and characteristic of the fermented soybean meal were analyzed and shown in Table 8. In addition, the components and characteristics of the raw soybean meal were analyzed and shown in Table 7.

[0190] In Table 7 and Table 8, the lactic acid concentration is represented by the amount of lactic acid comprised in the fermentation raw material or fermented product as w/w %. The KOH solubility means w/w % of quantifying the amount of the crude protein after extracting the fermentation raw material or fermented product with 0.2% KOH and dividing it by the total crude protein content. This is used as an index for the content of protein which can be absorbed in small intestines. The pepsin digestion rate is the value calculated by the following equation, after adding the fermentation raw material or fermented product to 0.2% pepsin hydrochloric acid solution and digesting for 16 hours in a 45° C. constant-temperature water bath and counting the crude protein content of undigested products.

Pepsin digestion rate (%)=(A−B)/A×100   [Equation 1]

[0191] (A: Crude protein content (g),

[0192] B: Crude protein content in undigested products (g))

[0193] Example 4-2: Preparation of Fermented Soybean Meal using Raw Soybean Meal (2)

[0194] With the same method except for using Enterococcus faecium SLB120 (KCTC12868BP), instead of Enterococcus faecium SLB130 (KCTC13566BP) used in Example 4-1, fermented soybean meal was prepared. Then, it was dried in a tray drier of 50° C. temperature, and the components and characteristics of fermented soybean meal were analyzed and shown in Table 8. In addition, the components and characteristics of raw soybean meal used as a fermentation raw material were analyzed and shown in Table 7.

EXAMPLE 5

Preparation of Fermented Soybean Meal using Residual Soybean Meal

Example 5-1: Preparation of Fermented Soybean Meal using Residual Soybean Meal (1)

[0195] By using Enterococcus faecium SLB130 (KCTC13566BP), with the substantially same method with Example 4, fermented soybean meal was prepared, but the residual soybean meal extracted with an extraction solvent was used instead of the raw soybean meal of Example 4 as a fermentation raw material to prepare fermented soybean meal. Then, it was dried in a tray drier of 50° C. temperature, and the components and characteristics of fermented soybean meal were analyzed and shown in Table 8. In addition, the components and characteristics of residual soybean meal used as a fermentation raw material were analyzed and shown in Table 7.

[0196] Example 5-2: Preparation of Fermented Soybean Meal using Residual Soybean Meal (2)

[0197] With the same method except for using Enterococcus faecium SLB120 (KCTC12868BP), instead of Enterococcus faecium SLB130 (KCTC13566BP) used in Example 5-1, fermented soybean meal was prepared by using the residual soybean meal obtained in Example 2 as a fermentation raw material. Then, it was dried in a tray drier of 50° C. temperature, and the components and characteristics of fermented soybean meal were analyzed and shown in Table 8.

EXAMPLE 6

Preparation of Fermented Soybean Meal using Mixed Soybean Meal

Example 6-1: Preparation of Fermented Soybean Meal using Mixed Soybean Meal (1)

[0198] By using Enterococcus faecium SLB130 (KCTC13566BP), with the substantially same method with Example 4, fermented soybean meal was prepared, but the mixed soybean meal raw material in which the raw soybean meal of Example 1 and the residual soybean meal of Example 2 were mixed at a weight ratio of 0.7:1 was used instead of the raw soybean meal of Example 4 as a fermentation raw material to prepare fermented soybean meal. Then, it was dried in a tray drier of 50° C. temperature, and the components and characteristics of fermented soybean meal were analyzed and shown in Table 8. In addition, the components and characteristics of the mixed soybean meal raw material used as a fermentation raw material were analyzed and shown in Table 7.

Example 6-2: Preparation of Fermented Soybean Meal using Mixed Soybean Meal (2)

[0199] With the same method except for using Enterococcus faecium SLB120 (KCTC12868BP), instead of Enterococcus faecium SLB130 (KCTC13566BP) used in Example 6, fermented soybean meal was prepared by using the mixed soybean meal raw material in which the raw soybean meal and the residual soybean meal were mixed at a weight ratio of 0.7:1 a fermentation raw material. Then, it was dried in a tray drier of 50° C. temperature, and the components and characteristics of fermented soybean meal were analyzed and shown in Table 8. In addition, the components and characteristics of the mixed raw material used as a fermentation raw material were analyzed and shown in Table 7.

TABLE-US-00007 TABLE 7 Item Raw Residual soybean soybean Mixed raw meal meal material Moisture content (w/w %) 12 65 40 pH 6.5 6.7 6.6 Crude protein content (w/w %) 46 30 38 Trypsin inhibitor (mg/g) 8.0 2.1 4.5 Beta-conglycininin (ppm) 70,000 65,000 68,000 Lactic acid concentration (w/w %) 0 0 0 Indigestible oligosaccharide 1.70 0.6 1.1 content (w/w %) KOH solubility (w/w %) 80 82 81 Pepsin digestion rate (w/w %) 82.7 85 83 Lactic acid bacteria viable cell ND ND ND (cfu/g) General bacteria (cfu/g) 5.4 × 10.sup.5 0.5 × 10.sup.5 4.8 × 10.sup.5

TABLE-US-00008 TABLE 8 Example Example Example Example Example Example Item 4-2 5-2 6-2 4-1 5-1 6-1 Moisture content 10 10 10 10 10 10 (w/w %) pH 6.0 5.2 5.7 5.8 5.1 5.4 Crude protein content 49 56 51 50 60 53 (w/w %) Trypsin inhibitor 3.5 1.2 2.1 1.1 0.8 1.0 (mg/g) Beta-conglycininin 35,000 640 1,500 620 180 420 (ppm) Lactic acid 3.4 3.9 3.7 3.8 5.1 4.6 concentration (w/w %) Indigestible 0.15 0.07 0.12 0.05 0.03 0.04 oligosaccharide content (w/w %) KOH solubility 70.1 70.7 70.4 73.0 77.4 76.4 (w/w %) Pepsin digestion rate 88.3 94.7 92.1 94.2 97.7 95.5 (w/w %) Lactic acid bacteria 1.1 × 10{circumflex over ( )}8 1.4 × 10{circumflex over ( )}8 1.2 × 10{circumflex over ( )}8 2.1 × 10{circumflex over ( )}8 3.8 × 10{circumflex over ( )}9 2.3 × 10{circumflex over ( )}8 viable cell (cfu/g) General bacteria 5.8 × 10{circumflex over ( )}4 5.4 × 10{circumflex over ( )}4 5.6 × 10{circumflex over ( )}4 3.4 × 10{circumflex over ( )}4 3.1 × 10{circumflex over ( )}3 1.2 × 10{circumflex over ( )}4 (cfu/g)

[0200] As the result of the experiment, it could be seen that in the soybean meal fermented product prepared according to the present invention, the crude protein content was increased, and the content of trypsin inhibitor, beta-conglycinin and indigestible oligosaccharides was significantly decreased, and the lactic acid concentration was increased, compared to the raw soybean meal.

[0201] The above result showed that in the fermented soybean meal prepared by the method for preparation of the present invention, the efficacy as fermented soybean meal such as further improved removal of anti-nutritional factors and increase of pepsin digestion rate, etc. was significantly increased, compared to the raw soybean meal, and sufficient component changes and characteristic modification occurred.

[0202] In addition, the crude protein content of the fermented soybean meal prepared by using SLB130 strain was higher than the fermented soybean meal prepared by using SLB120 strain, and the content of anti-nutritional factors such as indigestible oligosaccharides, trypsin inhibitors, beta-conglycinin, etc. was significantly reduced. In addition, the lactic acid concentration was increased and thereby the growth of contaminant was inhibited and the cell number of general bacteria was reduced.

[0203] For example, in case of Example 5 which used the residual soybean meal as a fermentation raw material, in the fermented soybean meal prepared by SLB130 strain (Example 5-1) compared to the fermented soybean meal using SLB120 strain (Example 5-2), the crude protein content was increased from 56% (w/w) to 60% (w/w) and the representative anti-nutritional factor, trypsin inhibitor was decreased from 1.2 mg/g to 0.8 mg/g in a considerable ratio (30% or more). Also, the content of beta-conglycinin was decreased from 640 ppm to 180 ppm by 50% or more. On the other hand, the indigestible oligosaccharides in soybean meal which played a harmful role by causing flatulence to livestock were significantly reduced from 0.7 mg/g to 0.3 mg/g. In addition, the lactic acid concentration was increased from 3.9% to 5.1%, and thereby the growth of contaminant was inhibited, and thus it was shown that the cell number of general bacteria was lowered.

[0204] For example, in case of Example 6 which used the mixed soybean meal as a fermentation raw material, in the fermented soybean meal prepared by SLB130 strain (Example 6-1) compared to the fermented soybean meal using SLB120 strain (Example 6-2), the crude protein content was increased from 51% (w/w) to 53% (w/w) and the representative anti-nutritional factor, trypsin inhibitor was decreased from 2.1 mg/g to 1.0 mg/g in a considerable ratio (50% or more). Also, the content of beta-conglycinin was decreased from 1,500 ppm to 420 ppm by 50% or more. On the other hand, the indigestible oligosaccharides in soybean meal which played a harmful role by causing flatulence to livestock were significantly reduced from 0.12 mg/g to 0.04 mg/g by 60% or more. In addition, the lactic acid concentration was increased from 3.7% to 4.6%, and thereby the growth of contaminant was inhibited, and thus it was shown that the cell number of general bacteria was lowered.

[0205] The above result showed that the fermented soybean meal prepared by the new lactic acid bacterium of the present invention, Enterococcus faecium SLB130 strain significantly increased the efficacy as fermented soybean meal such as further improved removal of anti-nutritional factors and increase of pepsin digestion rate, etc. than the fermented soybean meal prepared by the conventional Enterococcus faecium SLB120 strain, and sufficient component changes and characteristic modification occurred.

EXAMPLE 7

SDS-PAGE Analysis

7-1: Qualitative Analysis

[0206] To confirm the degree of increasing the digestion rate as protein in soybean meal was decomposed finely and decomposing beta-conglycinin and trypsin inhibitor, protein among anti-nutritional factors by the fermentation process, lg of each sample of raw soybean meal before fermentation (A), fermented soybean meal of Example 4-2 (B), fermented soybean meal of Example 5-1 (C, D, E) and fermented soybean meal of Example 4-1 (G) was added to 3m1 lysis buffer (urea 7M, thiourea 2M, CAHAPS 4%, DTT 40mM), and after voltexing at a maximum speed for 5 min and then heating at 95° C. for 5 min and centrifuging at 10,000 g for 10 min and then collecting supernatant, the concentration of protein was measured.

[0207] 20 ul of the same amount of protein in 12% TGX gel (Biorad) was added into a well and the degree of decomposition of protein was compared by SDS -PAGE. The result was shown in FIG. 1. In FIG. 1, A is the result of SDS-PAGE analysis result of the protein sample of the raw soybean meal before fermentation, and B is the result of SDS-PAGE analysis of the protein sample of the fermented soybean meal (Example 4-2) fermented by using SLB120, and C to E are the results of SDS-PAGE analysis of the protein sample of the fermented soybean meal (Example 5-1) in which the residual soybean meal was fermented by using SLB130, and F is a size marker, and G is the result of SDS-PAGE analysis of the protein sample of the fermented soybean meal (Example 4-1) in which the raw soybean meal was fermented by using SLB130. F of FIG. 1 is a size marker and each line represents the sizes of 250, 150, 100, 75, 50, 37, 25, 20, 15, and 10 kD in order from the top.

[0208] As the result, it could be seen that the degree of protein decomposition was increased in case of Example 4-1, and it could be seen that 30 kD or more of protein was mostly decomposed and the peptization of 30 kD or more was the highest in case of Example 5-1. Thus, it could be seen that through the fermentation process, the big size of protein in soybean meal was decomposed finely and the anti-nutritional protein was decomposed and thereby the digestion rate could be increased. In addition, the degree of decomposition in case of fermentation with SBL130 was higher than the case of fermentation with SLB120.

7-2: Quantitative Analysis

[0209] To investigate the degree of decomposition of protein in soybean meal by the fermentation process, the distribution of molecular weights of protein in the raw soybean meal before fermentation and the soybean meal after fermentation was measured. The result was shown in Table 9. The protein content of the following Table 9 means the weight percent of protein having the corresponding molecular weight range based on 100% by weight of the total protein in fermented soybean meal.

TABLE-US-00009 TABLE 9 Raw soybean Exam- Exam- Exam- Exam- Molecular weight meal ple5-1 ple6-1 ple4-1 ple4-2 Less than 25 kD 24 97 51 34 28 25 or more to less 45 2 40 50 46 than 50 kD 50 kD or more 31 1 9 16 26

[0210] As a result, the content of high molecule protein of molecular weight 50 kD or more was decreased and the content of protein less than 50 kD was relatively increased through the fermentation process. In particular, it could be confirmed that the content of protein less than 25 kD was particularly increased. In other words, it could be seen that the percentage of low molecule protein was increased than that before fermentation through the fermentation process. In addition, it could be confirmed that the degree of decomposition of protein in case of fermentation with SBL130 was higher than that in case of fermentation with SLB120.