KR20150089266A - Method for producing feed additive for livestock having antibacterial activity through solid state fermentation using Temphe and Bacillus sp. strain and its feed additive - Google Patents

Abstract

The present invention relates to a manufacturing method of a feed additive having antibacterial activity and a feed additive manufactured by the method, which comprises a step of increasing biomass of a strain by adding a mixture of a tempeh strain and a Bacillus sp. strain to grain and by solid state fermentation. No antibiotic, high-quality eco-friendly brand, and organic livestock product with improved productivity in farm can be provided for consumers, using a feed additive of the present invention.

Description

TECHNICAL FIELD The present invention relates to a method for preparing a feed additive for livestock having antimicrobial activity by solid phase fermentation using a Staphylococcus aureus strain and a Bacillus subtilis strain, and a feed additive for use in the feed additive for a livestock having antibacterial activity using a solid state fermentation using Temphe and Bacillus sp. strain and its feed additive}

The present invention relates to a method for preparing a feed additive having antimicrobial activity comprising adding a mixture of a temphe strain and a Bacillus sp. Strain to cereals and fermenting the same to increase the biomass of the strain, ≪ / RTI >

Conventional feed additives have been prepared by containing antibiotics or antimicrobial agents, which are synthetic chemicals, for the purpose of improving the growth rate of livestock, increasing feed efficiency and preventing diseases. However, the use of these feed additives in livestock feeds is a drug abuse of livestock, which results in a decrease in the immunity of the livestock to the disease and an increase in resistance to antibiotics, . In addition, drugs such as antibiotics remain in the body of livestock, so the health of those who ingest them may also be threatened. Therefore, in the animal industry, the research and development of efficacy and the research of candidates with safety that can substitute antibiotics and antimicrobial agents harmless to livestock and human beings for both consumers and producers are being conducted.

Tempe is made by fermenting soybeans and it is a representative fermented food in Indonesia. In traditional manufacturing method, beans are soaked in water, put in a bamboo basket, stepped on foot, peeled for about 1 hour in boiling water, And fermented in the fermentation chamber for 2 to 3 days.

Common probiotic fermentation methods include solid state fermentations (SSF) and liquid state fermentations (LSF), and there are differences in culture system and culture conditions depending on the fermentation type. Solid phase fermentation processes have important effects on microbial growth and product formation due to environmental factors such as moisture content, water activity, temperature, pH, oxygen level, nutrient and product concentration. In general, the water content in the solid phase fermentation process is 30-75% wide, and the optimal moisture content maximizes production of metabolites such as enzymes and organic acids. Water activity affects biomass development, metabolic reactions, and mass transfer processes, and its activity depends on the physical structure and chemical properties of the material. In addition, the increase in temperature during solid phase fermentation affects spore formation, growth, and formation of metabolites. Solid phase fermentation uses traditional fermentation starters such as Koji (Japan) and LAGI (Indonesia) to produce products with enhanced values such as enzymes, organic acids, biochemical pesticides, biofuels and flavors as well as bioremediation and biodegradation ), Decomposition of agricultural wastes, and so on.

The main fermenting bacteria in the preparation method of tempeh are Rhizopus R. oryzae , R. arrhizus, R. stolonifer and the like, R. oligosporus is an important fermenting fungus. Enzymes of Rizophus oligo sporose (hereinafter referred to as RO) decompose soybean tissue to make the tissue soft and viscous, reduce fat by fermentation, increase protein ratio, To increase the production of free amino acids, especially to produce alanine, proline, methionine, methionine, leucine and the like. Vitamin B1 is slightly reduced during fermentation, but vitamin B2, vitamin B6, nicotinic acid, and pantothenic acid are increased during fermentation. In addition, isoflavone, which is abundantly contained in soybean, is reported to be increased in content by RO fermentation process, and isoflavone content in general soybean and chongkukjang is higher than that of general soybean (Chung, Korean Journal of Food Science and Technology 44 (1) 2012).

In the present invention, a Bacillus sp. Strain producing a natural antibiotic substance and a hydrolytic enzyme as a substitute for antibiotics, a RO strain mixture having an immunological physiological activity and an excellent antioxidative ability, and a RO strain mixture are added to cereals and solid- To the diets, and to improve the nutrient digestibility, productivity and immunity of the livestock.

Korean Patent No. 1073986 discloses a novel Bacillus subtilis strain and a method of solid-phase fermentation of palm kernel seed pellet using the same. In Korean Patent Laid-Open No. 2011-0115188, Development of various combinations of cells and cells with high adaptability to disease improvement by maximizing the bioavailability of the jujuba extract "and Korean Patent Laid-Open Publication No. 2013-0036513 discloses a" probiotic agent with improved stability using jujube extract " There is no known method for preparing a feed additive for livestock having antimicrobial activity by solid-phase fermentation using a Staphylococcus aureus strain and a strain of Bacillus genus, and a feed additive therefor.

SUMMARY OF THE INVENTION The present invention has been made in view of the above needs, and the present invention has been made in view of the above circumstances, and it is an object of the present invention to provide a method for producing a natural antibiotic substance and a hydrolytic enzyme, Thus, a feed additive having antimicrobial activity was prepared by a solid phase culture system. As a result of administration of the feed additive to livestock, it was confirmed that the nutrient digestibility, productivity and immunity of livestock were improved.

In order to solve the above problems, the present invention provides a feed additive having antimicrobial activity, which comprises the step of adding a mixture of a temphe strain and a Bacillus sp. Strain to cereals and solid-phase fermentation to increase the biomass of the strain Of the present invention.

The present invention also provides a feed additive having antimicrobial activity produced by the above production method.

The present invention relates to a feed additive having an antimicrobial activity produced by a solid phase culture system using a strain of Bacillus sp. And a strain RO of Staphylococcus aureus as a starter. As a result of administering the feed additive to livestock, the nutrient digestibility and productivity And improved immunity. By using the feed additive of the present invention, it is possible to provide a non-antibiotic agent, a high-quality eco-friendly brand, and an organic livestock product to a consumer in addition to the productivity of a farmer.

FIG. 1 shows the results of investigation of the characteristics of a Bacillus subtilis strain suitable for a multi-SSF process, wherein A is a microscope image of a Bacillus subtilis strain, B is an antimicrobial activity against E. coli, and C is a test result of antimicrobial substance production per culture period.
Fig. 2 shows growth and pH changes of RO and Bacillus sp. Strains according to the culture method in a solid medium of grain. (A) and pH (B) according to the raw material for culture of RO strain alone, showing the number of spores and colonies (C) and pH (D) according to the culture medium during the co-culture of RO and Bacillus sp. (E) and pH (F) of the culture medium of Bacillus sp. Strain after primary fermentation of the strain.
FIG. 3 shows inhibitory activity against pathogenic microorganisms of RO and Bacillus sp. Strains. As a result, the inhibitory ability against E. coli K88 (FIG. A) and the inhibitory effect against Salmonella (FIG.

In order to achieve the object of the present invention, the present invention provides an antimicrobial activity comprising adding a mixture of an RO temphe strain and a Bacillus sp. Strain to cereals and solid-phase fermentation to increase the biomass of the strain A method for preparing a feed additive is provided.

The term "Tempe" is made by fermenting soybeans. It is a typical fermented food in Indonesia that has been eaten since ancient times, mainly in Java and Sumatra. In Indonesia, fermented foods are collectively referred to as fermented soybeans.

In the method according to one embodiment of the present invention, the mixing ratio of the Tempe and Bacillus strains may preferably be 1: 450 to 550 in terms of sfu (spore forming unit): cfu (colony forming unit) Preferably 1: 500, but is not limited thereto.

In the method according to one embodiment of the present invention, the Tempe strain may be a strain of the genus Rhizopus, preferably a strain of R. oryzae , R. arrhizus , R. stolonifer , or R. oligosporus , and most preferably R. oligosporus , but is not limited thereto.

In the method according to one embodiment of the present invention, the RO strain is an ATCC-approved strain Rhizopus oligosporus oligosporus NRRL2710 or Ragi, which is commercially available from Indonesia.

In the method according to an embodiment of the present invention, the Bacillus sp. Strain may be a strain having an ability to inhibit E. coli, preferably Bacillus subtilis , It does not.

In the method according to one embodiment of the present invention, the grain may be any one or more selected from the group consisting of rice, soybean meal, barley and corn, preferably soybean meal, mixed grain (soybean meal + corn + barley) And most preferably soybean meal, but is not limited thereto.

In the method according to an embodiment of the present invention, the solid-phase fermentation may be performed by adding a strain mixture to grains and culturing at 30 to 35 DEG C for 3 to 5 days, preferably (a) 40% < / RTI > for 30 minutes at < RTI ID = 0.0 > 80 C < / RTI > (b) culturing at 30 to 35 ° C for 3 to 5 days by adding a mixture of the Staphylococcus aureus strain and the Bacillus subtilis strain mixed at a ratio of 1: 450 to 550 to the boiled and cooled cereals by sfu: cfu; And drying the culture at a temperature of 40 to 60 ° C.

The present invention also provides a feed additive having antimicrobial activity produced by the above production method.

The feed additive according to one embodiment of the present invention may be 0.1-0.5%, preferably 0.15-0.3%, based on the total weight of the feed, but is not limited thereto.

The feed additive according to an embodiment of the present invention may be used for feeding piggy money, but is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Selection of strain

The Bacillus subtilis strain of the present invention was confirmed for its ability to inhibit E. coli and the production of antimicrobial substances at different culture stages to select a Bacillus subtilis strain suitable for solid phase fermentation (Fig. 1). The RO strain was an ATCC-approved strain Rhizopus oligosporus oligosporus NRRL2710 was purchased through a gene bank, and as a Tempe starter, Raghi purchased a commercial product from Indonesia.

2. Selection of liquid culture medium

The ATCC strain Rhizopus oligosporus ( Rhizopus oligosporus NRRL2710 and a Tempe starter in rice market in Indonesia were used, and as shown in Table 1, eight industrial liquid formulation ratios with different compositions of carbohydrate and protein and Potato Dextrose Broth as a control were used After culturing in a shaking incubator at 32 ° C and 170 rpm for 4 days, mycelium was recovered and the amount of mycelium was measured by an infrared spectrometer. The RO strain was inoculated with 2.0 × 10 ² sfu / ml spores and cultured for 2 days.

3. Mass production and yield of Tempe strain

The compounding ratio in Table 1 was determined by an industrial liquid culture medium and was determined by industrial mass culture using JA fermenter (KF-7L, Kobiotech, Korea, capacity 7L), temperature, stirring speed, air injection amount, Optimal conditions were checked in advance. In order to obtain a liquid-phase temp for the solid-phase fermentation, a large-scale culture was carried out using an industrial fermenter (FR-905, Kobiotech, Korea, capacity 5,000 L). The cultivation was carried out for 3 days (32 ° C, 100 rpm, air volume 1.0 vvm) after adding 3 L, 30 L, 300 L and 3,000 L of medium to the incubator. The mycelia were recovered by centrifugation (GSE-30-6-177, GEA Westfalia Separator, Germany) at 3,000 L at 4,000 rpm in 12,000 rpm to obtain the liquid phase temp for solid phase fermentation. (OPERON DFC-400CE, Operon Co., Korea) after freezing in an ultra-low temperature freezer at 75 ° C.

Eight industrial liquid formulation ratios with different carbohydrate and protein composition (Unit: g / 100 ml) division A B C D E F G H PDB Glucose 0.50 0.50 0.50 0.50 0.50 1.00 0.50 2.00 Corn starch 1.50 1.50 1.50 Soy flour 0.50 0.50 0.50 0.50 Yeast extract 0.10 0.10 0.10 0.10 0.10 0.10 0.30 0.30 KH ₂ PO ₄ 0.20 0.20 0.20 0.20 0.20 0.12 K ₂ HPO ₄ 0.06 MgSO ₄ 0.10 0.10 0.10 0.10 0.10 Soluble starch 1.50 1.50 0.50 0.40 Skim milk powder 0.50 0.50 0.30 CSL 0.40 1.50 molasses 1.00 0.50 Malt extract 0.30 Badge building 2.90 2.90 2.90 2.90 3.30 2.78 2.10 2.10 2.40

4. Solid phase fermentation culture

After heat treatment of rice, soybean meal, barley, corn and mixed grains (soybean meal + corn + barley) as a medium for solid fermentation with 40% moisture content at 80 ° C for 30 minutes, RO culture, RO culture, , And the cultivation of Bacillus sp. Strain after fermentation of RO strains. The fermentation was carried out at 32 ℃ for 3-4 days. Then, the spore count, Bacillus sp.

1) Single culture of RO strain

By a medium raw material for solid state fermentation with rice, a soybean meal, barley, corn and mixed grain (soybean meal + corn + barley) each moisture of 40%, heat-treated at 80 ℃ 30 minutes and then, RO spores 1.0 × 10 ⁴ (sfu / g ), Fermented at 32 ° C for 84 hours (3.5 days), and then subjected to hot air drying at 50 ° C.

2) Culture of RO and Bacillus sp.

By a medium raw material for solid state fermentation with rice, a soybean meal, barley, corn and mixed grain (soybean meal + corn + barley) each moisture of 40%, heat-treated at 80 ℃ 30 minutes and then, RO spores 1.0 × 10 ⁴ (sfu / g ) And 5.0 × 10 ⁶ (cfu / g) of Bacillus subtilis were inoculated simultaneously and fermented at 32 ° C for 84 hours (3.5 days) and then subjected to hot air drying at 50 ° C.

3) Cultivation of Bacillus subtilis strain after RO fermentation

By a medium raw material for solid state fermentation with rice, a soybean meal, barley, corn and mixed grain (soybean meal + corn + barley) each moisture of 40%, heat-treated at 80 ℃ 30 minutes and then, RO spores 1.0 × 10 ⁴ (sfu / g ), And after inoculation for 60 hours, 5.0 × 10 ⁶ (cfu / g) of Bacillus subtilis was inoculated and fermented again for 60 hours, followed by hot air drying at 50 ° C.

5. Enzyme activity measurement

As a result of the microbial analysis, the enzymes were analyzed using the metabolites of the dried cultures obtained from RO culture alone, RO culture, and Bacillus sp. For enzyme activity analysis, 10 g of the culture was centrifuged (10,000xg) to prepare a crude enzyme solution. Then, 0.1 ml of the enzyme solution was added to 1% soluble starch, and the color was induced with iodine solution to measure the absorbance at 670 nm 2004. α-Amylase, a test method for starch hydrolysis). The protease activity was determined by an enzyme reaction with a pH adjusted 0.6% casein solution as a substrate in a crude enzyme solution at 40 ° C for 20 minutes, and then the amount of the resulting folin non-peptidic substance was measured by the Folin colorimetric method . The enzyme activity unit was 1 unit (1 unit) of enzyme solution at 37 ° C and 1 μg of tyrosine per minute (Feed process, 2004. Acid Protease, Method 2 of protein digestibility test).

6. Vitamin activity measurement

As a result of the microbial analysis, vitamins were analyzed as metabolites of dried cultures obtained from single culture of Rizophus Oligospressus strain, RO culture, and Bacillus sp. Strain, which have the most culture adaptability and effect. For vitamin activity, vitamin E and vitamin B12 (cyanocobalamin) were analyzed according to the vitamin analysis method of Food Code (2005, p 367-368, p 383-385, Korea FDA).

7. Superoxide dismutase (SOD) activity measurement

The sample was reacted with a Tris-HCl buffer (pH 7.4) solution and hematoxylin in accordance with the method of Martin et al. (1987, Arch Biochem Biophys 255: 329-336) and the absorbance was measured at 560 nm. Activity was calculated by the enzyme activity (%) = 100 - [(control OD-sample OD) / (control OD) × 0.5].

8. Measurement of activity of isoflavones

To analyze the content of isoflavone, soybean meal fermentation broth was used as a sample. After 0.5 g of the dried culture was hydrolyzed with 1N HCl to convert the isoflavone glycoside to aglycone, the filtrate was centrifuged to remove the filtrate. We requested HPLC analysis from Dankook University.

9. Inhibition of pathogenic microorganisms

The disk diffusion method was used to measure the inhibitory activity of pathogenic microorganisms of RO and Bacillus subtilis strains.

10. Test animals and test design

[Landrace × Yorkshire × Duroc] Weighed 125 dogs were weighed and weighed 7.51 ± 0.73 kg at the start of the test. The experimental design was carried out with 5 kinds of basic diet, basic diet + Sunbeo Tempe 0.15%, basic diet + Sunbeo Tempe 0.3%, basic diet + Indonesian Tempe 0.15%, basic diet + Indonesian Tempe 0.3% , And 5 randomly placed randomly.

 11. Test feed and specification management

The test was conducted at Dankook University test farm. The test feeds were NRC (1998) According to the requirement, corn - soybean - based flour was prepared as a free vegetarian diet, and the water was adjusted to be eaten freely using an automatic water dispenser.

12. Daily gain, daily feed intake and feed efficiency

Body weight was measured by treatments at the start, at 2 weeks and at the end of 5 weeks. The feed intake was calculated by subtracting the remaining amount from the feed intake during body weight measurement. The feed efficiency was calculated by dividing the body weight gain by the feed intake.

13. Nutrient digestibility

Nutrient digestibility was obtained by adding 0.2% of chromium oxide (Cr ₂ O ₃ ) as a labeling substance at the end of 2 weeks and at the end of 5 weeks. The collected samples were dried in a dryer at 60 ° C for 72 hours and then pulverized using a Willy mill. Cr mixed with the general components of the feed and labeling was analyzed according to the method of AOAC (1995).

14. Blood characteristics

Blood samples were collected at 8 weeks after initiation and at the end of 5 weeks, and 2 ml of blood was collected from each jugular vein using a K3EDTA vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) Red blood cells, white blood cells and lymphocytes were examined using a blood analyzer (ADVID 120, Bayer, USA). Serum biochemical tests were performed by collecting 5 ml of blood from a jugular vein using a vacuum tube (Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ) and centrifuging at 3,000 rpm for 15 minutes at 4 ° C. The content of IgG in the separated serum was analyzed using a nephelometer (Behring, Germany) analyzer using a nephelometry method.

15. Microbial composition in the minute

Was collected in the swine was then frozen at the selected processing distinguished 8 at the end (5 weeks), harvesting the minute method drink anus, -20 ℃ to laboratory, homogenized and suspended in a sterile physiological saline solution after 10 ^{- 3} to 10 ^-7, and used as a sample for measuring viable cell count. MRS medium (de Man, Rogosa and Sharpe agar) was used for Lactobacillus and MacConkey agar (Difco, USA) was used for E. coli to measure the lactobacilli and E. coli bacteria in the feces After incubation for 38 hours at < RTI ID = 0.0 >

16. Diarrhea index

The diarrhea index was measured daily from the onset to 2 weeks, with the following exponents (1: firm / dry pellet, 2: firm / defecated, 3: soft / Not forming, 5; liquid).

17. Statistical processing

All data were analyzed by Duncan's multiple test (Duncan, 1955) using a general linear model of SAS (1996).

Example 1. Solid phase fermentation using RO and Bacillus sp.

end. RO culture alone culture

For the selection of grain for solid phase fermentation, solid phase fermentation was carried out with seven treatments of soybean meal, corn, barley, soybean meal + corn, corn + barley, soybean meal + barley, soybean meal + corn + barley. As a result, soybean meal, barley, Grain raw materials including rice were selected as cereals (soybean meal + corn + barley) and grain used as medium of Tempe starter in Indonesia. Cultivation was carried out by three methods: single culture of RO strain, simultaneous culture of RO and Bacillus sp. Strain, and culture of Bacillus sp. Strain after primary RO fermentation. As a result of RO culture alone, after the incubation for 84 hours, rice 7.0 × 10 ⁵ , soybean meal 1.0 × 10 ⁶ , barley 3.0 × 10 ⁷ , corn 5.0 × 10 ⁵ , mixed grain (soybean meal + corn + barley) 1.0 × 10 ⁵ (sfu / g), indicating that the number of spores of RO was significantly higher than that of other grains (Fig. 2A and Table 2). As a result of measuring the pH after 84 hours of incubation, the initial pH of the medium was increased to 7.66 and 7.37 at 6.35 and 6.30 in soybean meal and mixed grain (soybean meal + corn + barley), respectively, but not in other grain materials 2B and Table 2).

Spore Factor and pH Change by Culturing Single Culture of RO Strain in Solid Medium 0 hr 36 hr 60 hr 84 hr dry grain sfu / g pH sfu / g pH sfu / g pH sfu / g pH sfu / g pH rice 1.0x10 ⁴ 5.70 1.6x10 ⁶ 5.11 1.3x10 ⁶ 5.25 7.0x10 ⁵ 5.27 8.0x10 ⁵ 5.30 Soybean meal 1.0x10 ⁴ 6.35 1.2x10 ⁶ 6.76 6.0x10 ⁵ 7.37 1.0 x 10 ⁶ 7.66 1.0x10 ⁵ 7.16 barley 1.0x10 ⁴ 5.85 1.2x10 ⁷ 5.93 2.8 x 10 ⁷ 5.67 3.0x10 ⁷ 5.52 8.0x10 ⁶ 6.68 corn 1.0x10 ⁴ 6.06 1.0 x 10 ⁶ 5.29 4.0x10 ⁵ 4.42 5.0x10 ⁵ 4.32 1.0x10 ⁵ 5.96 Mixed grain
(Soybean meal +
Corn +
barley) 1.0x10 ⁴ 6.30 5.0x10 ⁶ 6.65 4.5x10 ⁵ 7.25 1.0x10 ⁵ 7.37 1.0x10 ⁵ 6.74

I. Culture of RO and Bacillus sp.

RO culture and Bacillus sp. Strain were found to be 2.3 × 10 ⁶ and 1.6 × 10 ⁶ sfu / g (spore forming units per gram) for barley and corn, respectively, for RO spore culture after 84 hours It was, which was higher than the rice pojasu 1.0 × 10 ^5, soybean meal 1.0 × 10 ^5, a mixed grain (corn soybean meal + + ^{barley) 5.0 × 10 5 (sfu /} g).

After culturing for 84 hours, the number of bacteria in the Bacillus was 5.0 × 10 ^{9 for} soybean meal, 3.2 × 10 ^{9 for} mixed cereals (soybean meal + corn + barley), 2.3 × 10 ^{9 for} barley, 1.8 × 10 ^{9 for} rice, 1.5 × 10 ⁹ (cfu / (Cfu / g) of corn, and the number of cereals was 1.8 × 10 ⁹ , mixed cereals (soybean meal + corn + barley) were 3.4 × 10 ⁹ , barley 1.6 × 10 ⁹ , rice 1.7 × 10 ⁹ , and corn 6.8 × 10 ⁸ (Figure 2C and Table 3). As a result of measuring the pH after culturing for 84 hours, the medium raw material was increased from 6.35 to 7.68 and 6.30 to 7.62 in soybean meal and mixed grain (soybean meal + corn + barley), but not in other grain raw materials Table 3).

Spore Factor of RO and Bacillus in Bacillus sp. Cultured with simultaneous mixing of RO and Bacillus sp. 0 hr 36 hr 60 hr 84 hr dry Strain grain sfu / g pH sfu / g pH sfu / g pH sfu / g pH sfu / g pH

RO rice 1.0x10 ⁴ 5.70 1.2x10 ⁵ 6.17 1.0x10 ⁵ 6.05 1.0x10 ⁵ 5.75 1.0x10 ⁵ 6.22 Soybean meal 1.0x10 ⁴ 6.35 1.0x10 ⁴ 7.03 1.0x10 ⁵ 7.36 1.0x10 ⁵ 7.68 5.0x10 ⁴ 6.94 barley 1.0x10 ⁴ 5.85 1.0 x 10 ⁶ 6.05 3.0x10 ⁶ 5.91 2.3x10 ⁶ 5.88 8.0x10 ⁵ 6.70 corn 1.0x10 ⁴ 6.06 1.1x10 ⁶ 5.02 1.1x10 ⁶ 4.62 1.6x10 ⁶ 4.59 1.0x10 ⁵ 6.55 Mixed grain
(Soybean meal + corn + barley) 1.0x10 ⁴ 6.30 5.0x10 ⁴ 6.89 1.0x10 ⁵ 7.48 5.0x10 ⁵ 7.62 1.0x10 ⁵ 6.64

Bacillus genus 0 hr 36 hr  60 hr 84 hr dry grain cfu / g pH cfu / g pH cfu / g pH cfu / g pH cfu / g pH rice 5.0x10 ⁶ 5.70 1.6x10 ⁹ 6.17 1.6 × 10 ⁹ 7.05 1.8x10 ⁹ 5.75 1.7x10 ⁹ 6.22 Soybean meal 5.0x10 ⁶ 6.35 7.3x10 ⁹ 7.03 4.5 × 10 ⁹ 7.36 5.0x10 ⁹ 7.68 1.8x10 ⁹ 6.94 barley 5.0x10 ⁶ 5.85 1.6x10 ⁹ 6.05 2.2 x 10 ⁹ 5.91 2.3x10 ⁹ 5.88 1.6x10 ⁹ 6.70 corn 5.0x10 ⁶ 6.06 1.0x10 ⁹ 5.02 9.0 × 10 ⁸ 4.62 1.5x10 ⁹ 4.59 6.0x10 ⁸ 6.55 Mixed grain
(Soybean meal + corn + barley) 5.0x10 ⁶ 6.30 3.8x10 ⁹ 6.89 2.3 × 10 ⁹ 7.48 3.2x10 ⁹ 7.62 3.4 x ^{10 9} 6.64

All. Cultivation of Bacillus subtilis strain after primary fermentation of RO strain

RO strains were cultured for 60 hours and then cultured for another 60 hours. As a result, the medium was found to be 1.1 × 10 ⁷ in barley, rice and corn, 1.0 x ^{10 6} , and 1.0 x 10 ⁶ (sfu / g), indicating higher number of spores than the other grain media (Fig. 2E and Table 4). As a result of culturing Bacillus sp. Strain after primary fermentation of RO strain, in all experiments except mixed grain, the number of Bacillus spp. Was 7.3 × 10 ⁹ , the mixed grain (soybean meal + corn + barley) was 3.8 × 10 ⁹ , 1.6 × 10 ⁹ , 1.6 × 10 ⁹ , and 1.0 × 10 ⁹ cfu / g of corn (FIG. 2E and Table 4), respectively. As a result of pH measurement after 84 hours of incubation, the soybean meal and mixed grains (soybean meal + corn + barley) increased from 6.35 to 7.68 and 6.30 to 7.62, respectively (Figs. 2F and 4).

Spore counts of RO and Bacillus spp. And pH after secondary fermentation with Bacillus sp. Strain after primary fermentation of RO strain in solid medium with grain 0 hr 36 hr 60 hr dry Strain grain sfu / g pH sfu / g pH sfu / g pH sfu / g pH

RO
Primary fermentation rice 1.3x10 ⁶ 5.70 2.0x10 ⁶ 6.17 3.0x10 ⁶ 6.05 1.0 x 10 ⁶ 6.22 Soybean meal 6.0x10 ⁵ 6.35 1.0x10 ⁵ 7.03 1.0x10 ⁵ 7.36 1.0x10 ⁵ 6.94 barley 2.8 x 10 ⁷ 5.85 3.1x10 ⁷ 6.05 3.0x10 ⁷ 5.91 1.1x10 ⁷ 6.70 corn 4.0x10 ⁵ 6.06 8.1x10 ⁵ 5.02 1.3x10 ⁶ 4.62 1.0 x 10 ⁶ 6.55 Mixed grain
(Soybean meal + corn + barley) 4.5x10 ⁵ 6.30 1.0x10 ⁵ 6.89 1.0x10 ⁵ 7.48 8.0x10 ⁴ 6.64

Bacillus genus culture 0 hr 36 hr 60 hr dry grain cfu / g pH cfu / g pH cfu / g pH cfu / g pH rice 5.0x10 ⁶ 5.70 3.0x10 ⁷ 6.17 1.0 x 10 ⁷ 7.05 1.2 x ^{10 8} 6.22 Soybean meal 5.0x10 ⁶ 6.35 2.9x10 ⁹ 7.03 2.4 × 10 ⁹ 7.36 4.0x10 ⁹ 6.94 barley 5.0x10 ⁶ 5.85 6.5 x ^{10 8} 6.05 5.6 x 10 ⁸ 5.91 7.0 x ^{10 8} 6.70 corn 5.0x10 ⁶ 6.06 2.0x10 ⁹ 5.02 1.6 × 10 ⁹ 4.62 6.5 x ^{10 8} 6.55 Mixed grain
(Soybean meal + corn + barley) 5.0x10 ⁶ 6.30 2.4x10 ⁹ 6.89 2.8 × 10 ⁹ 7.48 3.0x10 ⁹ 6.64

Example 2. Enzyme and Vitamin Assay of Dried Cultures Obtained from Single Culture of RO Strain and Simultaneous Mixture Culture of RO and Bacillus sp.

Enzyme and vitamin analysis of the dried cultures obtained from RO culture alone, RO and Bacillus sp. Cultures resulted in protease activity of 358.9 (u / ml) in RO culture alone, RO and Bacillus sp. g) and 427.3 (u / g), respectively, and the α-amylase activity was 49.5 (u / g) and 412.4 (u / g) . Vitamin E was 272.1 (ppm) and 343.0 (ppm), vitamin B12 was 29.0 (ppm) and 50.3 (ppm), and the content of superoxide dismutase (SOD) was 340.0 The total isoflavone content was 800.0 (㎍ / g) and 912.0 (㎍ / g), respectively (Table 5). RO and Bacillus strains showed 8.33 times of alpha-amylase, 1.73 times of vitamin B12, 1.52 times of superoxide dismutase (SOD), 1.26 times of vitamin E, 1.19 times of protease, 1.16 times of isoflavone (Table 5).

Enzyme and Vitamin Activity by Simultaneous Culture of RO Strain and Culture of RO and Bacillus sp. division unit RO
Culture alone RO culture and Bacillus sp. Protease
(Protease) u / g 358.9 427.3 Alpha-amylase
(? -amylase) u / g 49.5 412.4 Vitamin E ppm 272.1 343.0 Vitamin B12 ppm 29.0 50.3 Superoxide
Disutase (SOD) u / mg 340.0 520.0  Isoflavone μg / g 800.0 912.0

Example 3 Inhibition of Pathogenic Microorganisms of RO and Bacillus sp.

The Bacillus sp. Strain was inhibited in both E. coli and Salmonella typhimurium KCCM 40253, while the RO strain could not be inhibited in E. coli and Salmonella typhimurium KCCM 40253 (FIG. 3).

Example 4. Productivity according to the level of Tempe added in the feed of the present invention

)를 첨가한 사료를 사용하였다. 사료내 템페의 첨가가 이유자돈의 생산성에 미치는 영향을 표 6에 나타내었다. 0-2주차에 있어 사료섭취량은 기초사료 + 인도네시아 템페 0.15% 처리구가 기초사료 + 선바이오의 템페 0.3% 처리구보다 유의적으로 높게 나타났지만, 일당증체량과 사료효율에 있어서는 처리구간 유의적인 차이를 나타내지 않았다(P>0.05). 3-5주차에 있어 일당증체량은 기초사료 + 선바이오의 템페 0.15% 처리구가 기초사료 및 기초사료 + 인도네시아 템페 0.3% 처리구보다 높게 나타났다. 일당사료섭취량 및 사료효율에 있어서는 처리구간 유의적인 차이를 나타내지 않았다(P>0.05). 전체 시험기간 동안의 일당증체량 또한 기초사료 + 선바이오의 템페 0.15% 처리구가 기초사료 및 기초사료 + 인도네시아 템페 0.3% 처리구보다 유의적으로 가장 높게 나타났지만, 일당사료섭취량 및 사료효율은 처리구간 유의적인 차이를 나타내지 않았다(P>0.05). The sunbeam tempe used in this experiment was prepared by co-culturing RO and Bacillus strains of Example 1, and soybean meal was used for the experiment. The control diet consisted of basal diet and RAPRIMA

) Were added. Table 6 shows the effect of the addition of tempefos in the feed on the productivity of the weaned piglets. Feed intake in basal diet + 0.15% of Indonesian tempeh was significantly higher than that of basic diet + Sunbio Tempe 0.3% for 0-2 week, but there was no significant difference in daily gain and feed efficiency (P > 0.05). For the 3-5th week, the daily weight gain was higher in basic diet + Sunbeo's Tempe 0.15% than in basic diet and basic diet + Indonesian Tempe 0.3%. There was no significant difference in dietary intakes and feed efficiency between treatments (P> 0.05). Daily body weight gain during the whole test period was also significantly higher than that of the basic diet + basic diet + basic diet + 0.1% of Tempe 0.1% of Sunbio, compared with 0.3% of Indonesian tempeh diet. However, daily dietary intake and feed efficiency were significantly (P > 0.05).

Growth of weaned piglets according to the level of tempeh in feed Item Foundation
feed Basic diet +
Sunbeo's Tempe 0.15% Basic diet +
Sunbeo's Tempe 0.3% Basic diet +
Indonesia Tempe 0.15% Basic diet +
Indonesia Tempe 0.3% Standard
error 1 (0-2 weeks)   Daily gain (g) 309 332 311 320 303 15   Daily dietary intake (g) 388 ^ab 402 ^ab 361 ^b 409 ^a 369 ^ab 14   Feed efficiency 0.796 0.826 0.861 0.782 0.821 0.049 Second (3-5 weeks)   Daily gain (g) 619 ^bc 655 ^a 631 ^ab 637 ^ab 596 ^c 8   Daily dietary intake (g) 957 947 927 971 936 26   Feed efficiency 0.647 0.692 0.681 0.656 0.637 0.020 all
(1-5 weeks)   Daily gain (g) 495 ^bc 526 ^a 503 ^abc 510 ^ab 479 ^c 8   Daily dietary intake (g) 729 729 700 746 709 19   Feed efficiency 0.679 0.722 0.719 0.684 0.676 0.020 ^{a, b, c} The other subscripts in the same row have significant differences (P <0.05).

Example 5. Nutrient digestibility according to the level of Tempe added in the feed of Isoflavone

Table 7 shows the effect of the addition of tempefos in the feed on the nutrient digestibility of the weaned piglets. Nitrogen digestibility in the second week was higher in the order of basic diet + Sunbio supplemented with Sunbio Tempe, basic diet + Sunbio Tempe 0.3%, basic diet + Indonesian Tempe 0.3%, followed by Sunbio Tempe and Indonesia There was no significant difference between treatment and storage periods and energy digestibility (P> 0.05). Nitrogen digestibility at 5th week was higher than basal diet + Indonesian tempeh 0.15% and basic diets, and 0.15% of basal diet + sunbio and basic diet + Sunbio tempeh 0.3% 0.15% of basal diet supplemented with Sunbio Tempe + Sunbio and 0.3% of basic diet + Sunbio Tempe 0.3% were significantly higher than those of basal diet and basic feed + 0.3% of Indonesian tempeh. The digestibility of dry matter was significantly (P > 0.05). This test was conducted to evaluate the functional probiotics using Staphylococcus aureus and Antibacterial Bacillus strains. The productivity of weaned pork was 0.15% with Sunbio Tempe Probiotics during the entire test period. It was improved over basal diet and Indonesian tempeh additive treatment.

Nutrient digestibility of weaned piglets according to the level of tempeh in feed Item(%) Foundation
feed Basic diet +
Sunbeo's Tempe 0.15% Basic diet +
Sunbeo's Tempe 0.3% Basic diet +
Indonesia Tempe 0.15% Basic diet +
Indonesia Tempe 0.3% Standard
error 2 weeks building 80.29 82.66 82.08 82.93 81.50 1.05 nitrogen 82.17 ^b 86.94 ^a 85.93 ^a 85.83 ^a 84.18 ^ab 1.01 energy 81.45 83.04 83.09 83.29 81.86 0.97 5 weeks building 79.70 82.59 82.56 82.13 80.63 0.93 nitrogen 80.80 ^b 85.29 ^a 84.98 ^a 84.40 ^a 82.75 ^ab 1.07 energy 78.86 ^b 82.72 ^a 82.11 ^a 80.65 ^ab 79.29 ^b 0.93
^{a, b} The other subscripts in the same row have significant differences (P <0.05).

Example 6. Blood characteristics according to the level of Tempe added in the feed of the present invention

Table 8 shows the effects of the addition of tempex in the feed on the blood characteristics of the weaned piglets. At the end of the experiment, IgG content in blood was higher in Sunbio Tempe and Indonesia Tempe treatment than in the basal diet. In the Tempe treatment period, 0.15% of basic diet + Sunbio Tempe, 0.3% of basic diet + Feed + Sunbeo's Tempe 0.3%, basic feed + Indonesian Tempe 0.15%, followed by basic feed. During the whole test period, there was no significant difference in blood leukocyte, erythrocyte, and lymphocyte levels (P> 0.05).

Blood Characteristics of Piglets According to Level of Tempe Item Foundation
feed Basic diet +
Sunbeo's Tempe 0.15% Basic diet +
Sunbeo's Tempe 0.3% Basic diet +
Indonesia Tempe 0.15% Basic diet +
Indonesia Tempe 0.3% Standard
error 0 weeks Red blood cells, 10 ⁶ / 6.23 6.36 6.35 6.45 6.54 0.19 Leukocyte, 10 ³ / 12.81 12.93 13.19 11.81 12.12 0.88     Lymphocytes,% 50.1 51.9 51.1 50.9 50.6 2.9    Immunoglobulin (IgG), mg / dL 302.8 320.8 312.8 311.0 307.5 8.8 5 weeks Red blood cells, 10 ⁶ / 6.24 6.43 6.29 6.38 6.21 0.18 Leukocyte, 10 ³ / 16.85 16.87 17.03 17.49 17.43 0.72     Lymphocytes,% 57.0 58.5 57.3 57.3 58.6 3.1    Immunoglobulin (IgG), mg / dL 301.0 ^b 325.8 ^a 309.8 ^ab 308.3 ^ab 311.5 ^ab 6.8 ^{a, b} The other subscripts in the same row have significant differences (P <0.05).

Example 7. Microbial composition in the intramuscular phase according to the addition level of Tempe &lt; RTI ID = 0.0 &gt;

Table 9 shows the effect of the addition of tempefos in the feed on the microbial composition of intramuscular Ilefect. Lactobacillus and Escherichia coli did not show any significant difference in the treatments (P> 0.05). There was no difference in the number of coliform bacteria among the microbial counts in the intramuscular stage, however, the number of the bacteria was low.

Microbial composition in the mouth according to the level of Tempe Item, log ₁₀ cfu / g Foundation
feed Basic diet +
Sunbeo's Tempe 0.15% Basic diet +
Sunbeo's Tempe 0.3% Basic diet +
Indonesia Tempe 0.15% Basic diet +
Indonesia Tempe 0.3% Standard
error Lactobacillus 7.69 7.71 7.74 7.71 7.7 0.06 Escherichia coli 6.52 6.48 6.46 6.50 6.48 0.08

Example 8. Diarrhea index according to the addition level of Tempe &lt; RTI ID = 0.0 &gt;

Table 10 shows the effect of the addition of tempex in the feed on the diarrhea index of the weaned piglets. There was no statistically significant difference in the diarrhea index from 0 to 2 weeks (P> 0.05).

Diarrhea index according to the level of Tempe added in feed Item Foundation
feed Basic diet +
Sunbeo's Tempe 0.15% Basic diet +
Sunbeo's Tempe 0.3% Basic diet +
Indonesia Tempe 0.15% Basic diet +
Indonesia Tempe 0.3% Standard
error Fecal index 3.21 3.17 3.16 3.16 3.16 0.02 Excreta index: determined at 08:00 and 20:00, 1 (rigid, dry pellets) depending on the excrement scoring system; 2 (firm, defecation); 3 (soft, bodied bowels that maintain shape); 4 (soft, unable to form bowel movements); 5 (liquid)

Claims (12)

Adding a mixture of a temphe strain and a Bacillus sp. Strain to the grain and solid-phase fermentation to increase the biomass of the strain. [Claim 2] The method according to claim 1, wherein the mixing ratio of the Tempe and Bacillus strains is 1: 450-550 on the basis of sfu: cfu. The method according to claim 1, wherein the Tempe strain is a Rizopus sp. Strain. [Claim 5] The method according to claim 3, wherein the Tempe strain is a Rizopus oligosporus strain. The method according to claim 1, wherein the Bacillus sp. Strain is Bacillus subtilis . [Claim 3] The method according to claim 1, wherein the Bacillus sp. Strain has antibacterial activity against E. coli. The method according to claim 1, wherein the grain is at least one selected from the group consisting of rice, soybean, barley and corn. [Claim 3] The method according to claim 1, wherein the solid phase fermentation is performed by adding a strain mixture to a grain and culturing the mixture at 30 to 35 DEG C for 3 to 5 days. 9. The method of claim 8,
(a) boiling the so-called water and cooling it;
(b) wherein (a) the grain of boiled and cooled sfu steps: a cfu reference 1: The oligonucleotide separation tank Perth mixed at a ratio of 450 ~ 550 Spokane Russ (Rizopus oligosporus) strains and the Bacillus subtilis standing (Bacillus subtilis ), and culturing the cells at 30 to 35 DEG C for 3 to 5 days; And
(c) drying the culture of step (b) at a temperature of 40 to 60 ° C. A feed additive having antimicrobial activity produced by the method of any one of claims 1 to 9. The feed additive of claim 10, wherein the feed additive is added in an amount of 0.15 to 0.3% based on the total weight of the feed. 11. The feed additive of claim 10, wherein the feed additive is for feeding pigs.

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