BACILLUS SUBTILIS CJBS303 AND COMPOSITION COMPRISING SAME

Abstract

Provided are a Bacillus subtilis CJBS303 strain and use thereof. Also, provided are a strain having an effect of reducing livestock manure-derived odors, a composition and a microbial preparation, each including the strain, and a feed composition including the composition.

Claims

1. A Bacillus subtilis CJBS303 strain deposited with the Accession No. KCCM12435P.

2. The Bacillus subtilis CJBS303 strain of claim 1, wherein the strain has an effect of reducing livestock manure-derived odors.

3. The Bacillus subtilis CJBS303 strain of claim 1, wherein the strain reduces levels of one or more odorous gases selected from the group consisting of ammonia, hydrogen sulfide, methyl mercaptan, indoles, phenols, and volatile fatty acids (VOCs).

4. A composition comprising the Bacillus subtilis CJBS303 strain of claim 1, a culture of the strain, a concentrate of the culture, or a dry product of the concentrate.

5. The composition of claim 4, further comprising a cryoprotectant or an excipient.

6. The composition of claim 5, wherein the cryoprotectant is one or more selected from the group consisting of glycerol, trehalose, maltodextrin, skimmed milk, and starch.

7. A feed composition comprising the composition of claim 4.

8. The feed composition of claim 7, comprising 1.0×10.sup.7 cfu/kg to 1.0×10.sup.11 cfu/kg of the Bacillus subtilis CJBS303 strain, based on the total weight of the composition.

9. A microbial preparation for removing livestock odors, comprising the Bacillus subtilis CJBS303 strain of claim 1.

10. The microbial preparation of claim 6, wherein the microbial preparation reduces levels of one or more odorous gases selected from the group consisting of ammonia, hydrogen sulfide, methyl mercaptan, indoles, phenols, and volatile fatty acids (VOCs).

11. A method of reducing odors from manure of an individual, comprising administering, to the individual, the strain of claim 1.

12. A method of preparing a microbial preparation, comprising: culturing a Bacillus subtilis CJBS303 strain deposited with the Accession No. KCCM12435P; and mixing the recovered strain with an additive.

13. The method of claim 12, wherein the additive is a cryoprotectant, and the method further comprises freeze-drying the obtained mixture, after the mixing.

14. The method of claim 13, wherein the strain in a freeze-dried microbial preparation obtained by the freeze-drying is in a viable state.

15. The method of claim 12, wherein the microbial preparation comprises 1.0×10.sup.7 cfu/kg to 1.0×10.sup.11 cfu/kg of the Bacillus subtilis CJBS303 strain, based on the total weight of the microbial preparation.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0061] FIG. 1 shows results of evaluating ammonia consumption capacity according to one exemplary embodiment, wherein color development of culture media according to ammonia consumption was visually examined;

[0062] FIG. 2 shows results of quantitatively comparing ammonia consumption rates of CJ109, CJ251, CJ268, and CJBS303 strains which were cultured in ammonia-containing media;

[0063] FIG. 3 shows electron microscopy results of examining morphological characteristics of the CJBS303 strain;

[0064] FIG. 4 shows results of visually examining hemolysis of the CJBS303 strain;

[0065] FIG. 5 shows results of examining changes of ammonia generation from pig fecal slurries according to addition of the CJBS303 strain;

[0066] FIG. 6 shows results of examining changes in ammonia and hydrogen sulfide generation from manure according to feeding of the CJBS303 strain;

[0067] FIGS. 7A to 7E show effects of reducing odorous gases in a pig pen according to feeding of the CJBS303 strain, wherein FIGS. 7A, 7B, 7C, 7D, and 7E show results of examining complex odor levels in a pig pen, ammonia levels in the pig pen, hydrogen sulfide levels in the pig pen, volatile fatty acid (VOCs) levels in the pig pen, and methyl mercaptan levels in the pig pen over time, respectively; and

[0068] FIGS. 8A to 8D show effects of reducing odorous gases in a pig pen according to feeding of the CJBS303 strain, wherein FIGS. 8A, 8B, 8C, and 8D show results of comparing ammonia levels in a pig pen, hydrogen sulfide levels in the pig pen, methyl mercaptan levels in the pig pen, and complex odor levels in the pig pen, respectively.

MODE OF DISCLOSURE

[0069] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating the present disclosure, and the scope of the present disclosure is not limited to these exemplary embodiments.

EXAMPLE 1. ISOLATION AND IDENTIFICATION OF STRAIN HAVING EFFECT OF REDUCING LIVESTOCK MANURE-DERIVED ODORS

[0070] (1) Acquisition of Samples and Isolation of Strain

[0071] Samples such as soybean paste, red pepper paste, blocks of fermented soybeans, etc., which are traditional Korean sauces, were obtained, and the samples were serially diluted and then plated on a Brain Heart Infusion (BHI, Difco) solid medium containing 3% sodium chloride, followed by incubation at 37° C. for 24 hr. Strains isolated from each sample were grouped and separated according to colonies. The selected colonies were purified by transferring the colonies to a new medium over three times, and a total of 465 strains purified and cultured were stored in a medium, to which 20% glycerol was added, at a temperature below −70° C.

[0072] (2) Selection of Strain by Evaluation of Ammonia Consumption Capacity

[0073] To select strains that consume ammonia which is a main cause of odors, ammonia consumption capacity of the strains was evaluated. The ammonia consumption capacity was examined by a qualitative method of using a reagent (sulfanilic acid, N, N-Dimethyl-1-naphthylamine) that develops a color by reacting with nitrite resulting from oxidation of ammonia. In detail, ammonia-added broths (containing 4.95 g/L of (NH.sub.4).sub.2SO.sub.4, 8.82 g/L of K.sub.2HPO.sub.4, 1.1 ml/L of 1 M MgSO.sub.4 solution, 0.3 ml/L of 1 M CaCl.sub.2 solution, 0.5 ml/L of 30 mM FeSO.sub.4 solution, 0.04 ml/L of 50 mM CuSO.sub.4 solution, 0.7 g/L of NaH.sub.2PO.sub.4, and 12 ml/L of 5% (WN) Na.sub.2CO.sub.3 anhydrous) were prepared, and then each of the strains was seeded at a concentration of 0.01% in 10 ml of the prepared broth, followed by incubation at 30° C. for 14 days. Thereafter, each of the culture broths was centrifuged, and 1 ml of each supernatant was collected. 100 μl of the reagent that develops a color by reacting with nitrite was added thereto. Each culture supernatant, to which the reagent was added, was allowed to react at 25° C. for 10 min to induce color development, and then color development of the culture broth was observed. Meanwhile, in the following exemplary embodiments, Bacillus subtilis KCCM11143P disclosed in Korean Patent Publication No. 10-2012-0088436 was used as a standard strain.

[0074] As shown in FIG. 1, when nitrite is produced by consumption or oxidation of ammonia, the culture broth developed dark purple, and strains having ammonia consumption capacity were primarily selected, based on the color development. As a result, of 456 kinds of strains isolated from the samples, 4 kinds of strains having significant ammonia consumption capacity, specifically, CJ109, CJ251, CJ268, and CJBS303 were selected.

[0075] In addition, the ammonia consumption was quantitatively compared between 4 kinds of the strains primarily selected. Specifically, after seeding each of the strains at a concentration of 0.1% in a BHI liquid medium, the strain was incubated at 200 rpm and 37° C. for 15 hr to activate the strain. Each of the activated strains was seeded at a concentration of 1% in an ammonia-containing medium (containing 0.5 g/L of (NH.sub.4).sub.2SO.sub.4, 13.5 g/L of NaH.sub.2PO.sub.4, 0.7 g/L of K.sub.2HPO.sub.4, 0.1 g/L of MgSO.sub.4 .Math.7H.sub.2O, 0.18 g/L of CaCl.sub.2 .Math.2H.sub.2O, 0.5 g/L of NaHCO.sub.3, 0.014 g/L of FeCl.sub.3.Math.6H.sub.2O, 0.5 g/L of glucose), followed by incubation at 200 rpm and 37° C. for 6 hr. After the culture was completed, the culture broth was centrifuged, from which only the culture supernatant was recovered. The ammonia content in the initial medium and the residual amount of ammonia in the recovered culture supernatant were quantified, and ammonia consumption rates were calculated according to Equation 1.

Ammonia consumption rate (%)=(A−B/A)×100   [Equation 1]

[0076] (A: Initial Ammonia Content, B: Ammonia Content after Completing Culture)

[0077] As a result, as shown in FIG. 2, among 4 kinds of the strains primarily selected, the CJBS303 strain showed the highest ammonia consumption rate of 74.3%. Therefore, CJBS303, which has the highest ammonia consumption rate, was finally selected as a strain having an effect of reducing odors derived from livestock manure.

[0078] (3) Morphological and Biochemical Characterization

[0079] Morphological and biochemical characteristics of the CJBS303 strain were analyzed. Specifically, morphological characteristics of the strain were analyzed by Gram staining and electron microscopy, and the biochemical characteristics of the strain were analyzed by examining sugar fermentation patterns of the strain through an API 50 CHB system (biomerieux Vitek, Inc., France).

[0080] As a result, it was confirmed that the CJBS303 strain is a Gram-positive, rod-shaped bacterium, as shown in FIG. 3. As shown in Table 1, the CJBS303 strain was confirmed to exhibit sugar fermentation patterns similar to Bacillus subtilis .

TABLE-US-00001 TABLE 1 Sample Fermentation Sample Fermentation Control − Esculine + Glycerol + Salicine + Erythritol − Cellobiose + D-Arabinose − Maltose + L-Arabinose − Lactose + Ribose + Melibiose − D-Xylose + Saccharose + L-Xylose − T rehalose + Adonitol − Inuline − β Methyl-xyloside − Melezitose − Galactose − D-Raffinose + D-Glucose + Amidon − D-Fructose + Glycogene − D-Mannose + Xylitol − L-Sorbose − β Gentiobiose − Rhamnose − D-T uranose − Dulcitol − D-Lyxose − Inositol + D-T agatose − Mannitol + D-Fucose − Sorbitol + L-Fucose − α Methyl-D-mannoside − D-Arabitol − α Methyl-glucoside + L-Arabitol − N Acetyl glucosamine − Gluconate − Amygdaline + 2 keto-gluconate − Arbutine + 5 keto-gluconate −

[0081] (4) Identification of Strain

[0082] For identification of the strain, molecular phylogenetic analysis of a DNA sequence was performed. To this end, the gene of 16s rDNA was amplified using PCR premix (Bioneer, Korea) and universal primers of 27F (5′AGAGTTTGATCMTGGCTCAG 3′), and 1492R (5′ GGTTACCTTGTTACGACTT 3′). When the gene was amplified, the total reaction solution was set to 20 μl, and repeated 30 times in total under conditions of at 94° C. for 1 min, at 56° C. for 1 min, and at 72° C. for 1 min. The 16s rDNA sequence of CJBS303 strain was the same as SEQ ID NO: 1, which showed 99% homology with Bacillus subtilis. In the following exemplary embodiments, the isolated strain was named ‘Bacillus subtilis CJBS303’or ‘CJBS303 strain’ (Accession No. KCCM12435P).

[0083] EXAMPLE 2. EVALUATION OF SAFETY OF CJBS303 STRAIN

[0084] To confirm safety of the CJBS303 strain, hemolysis of the strain was evaluated. In detail, the CJBS303 strain was streaked on a blood agar plate (5% sheep blood, Hanil-KOMED, Korea), and then incubated at 37° C. for 24 hr. Thereafter, hemolysis on the cultured medium was visually observed.

[0085] As a result, as shown in FIG. 4, the CJBS303 strain showed no hemolysis, indicating safety of Bacillus subtilis CJBS303 strain.

[0086] EXAMPLE 3. EVALUATION OF EFFECT OF REDUCING LIVESTOCK MANURE ODOR OF CJBS303 STRAIN

[0087] (1) Effect of Reducing Odorous Gases in Fecal Slurry

[0088] The effect of reducing odorous gases in a pig fecal slurry of the CJBS303 strain was compared with that of the standard strain. In detail, after culturing the CJBS303 strain in a trypticase soy broth (TSB), 4 ml of a solution containing 1×10.sup.8 cfu/kg of the CJBS303 strain was added to 400 ml of pig manure slurry, which was incubated under shaking at 120 rpm and 39° C. under facultative aerobic conditions for a total of 48 hr. 24 hr and 48 hr from the time of incubation, the amount of ammonia gas was measured for a total of twice using a portable odorous gas monitor (MultiRAE). The total amount of ammonia was compared by summing the amount of gas measured twice for each treatment group.

[0089] As a result, as shown in FIG. 5, the total generation of ammonia gas was reduced by 41%, as compared with a non-CJBS303 strain-added control, indicating that the reducing effect was 4 times or higher, as compared with the standard strain.

[0090] (2) Effect of Reducing Manure-Derived Odorous Gases

[0091] A probiotic containing the CJBS303 strain was fed to pigs, and its effect on generation of manure-derived odorous gas was examined. A total of 32 heads of finishing pigs were divided into two groups, and a feeding experiment was performed. A group fed with an additive-free feed was set as a control, and the control feed was fed to finishing pigs of a total of 4 pens, four heads of finishing pigs each pen, for 5 weeks. Meanwhile, a group that fed with a feed containing 1×10.sup.9 cfu/kg of the CJBS303 strain in the feed was set as a treatment group, and the feed was fed to finishing pigs of a total of 4 pens, four heads of finishing pigs each pen, for 5 weeks. Manures derived from the control and treatment groups were sampled every week from week 2 after feeding, and the odorous gases generated therefrom were measured. The manures sampled from the control and treatment groups were mixed at a constant ratio (600 g of feces+1.2 L of 12.5% diluted urine), and then subdivided into 300 ml in a bottle, followed by stationary incubation at 39° C. under facultative aerobic conditions for a total of 42 hr. 12 hr, 18 hr, 36 hr, and 42 hr from the time of incubation, the amounts of generated gases, i.e., hydrogen sulfide and ammonia gas, were measured for a total of four times using a portable odorous gas monitor (MultiRAE). The manure culture experiment was conducted every week when sampling of manure was performed, and a reduction rate relative to the control was calculated by averaging the data until week 5.

[0092] As a result, as shown in Table 2 and FIG. 6, the treatment group was observed to have lower concentrations of ammonia and hydrogen sulfide gases than the control from week 2 after feeding, and it was confirmed that when the concentrations of the gases generated until week 5 were averaged and compared, the reduction rate of odorous gases according to feeding of the CJBS303 strain was 38.3% for ammonia and 88.9% for hydrogen sulfide.

TABLE-US-00002 TABLE 2 Ammonia (ppm) Hydrogen sulfide (ppm) Control CJBS303 0.1% Control CJBS303 0.1% Week 2 207 140 286 14 Week 3 278 152 166 22 Week 4 212 135 52 20 Week 5 180 113 91 10 Average 219 135 149 16 Reduction 38.30% 88.90% rate relative to control

[0093] (3) Effect of Reducing Odorous Gases in Pig Pen

[0094] A probiotic containing the CJBS303 strain was fed to pigs, and its effect on generation of odorous gases in a pig pen was examined. A total of 96 heads of 74-day-old finishing pigs were divided into two groups, and a feeding experiment was performed. A group fed with an additive-free feed was set as a control, and the control feed was fed to growing pigs of a total of 12 pens, four heads of growing pigs each pen, for 4 weeks. Meanwhile, a group that fed with a feed containing 1×10.sup.9 cfu/kg of the CJBS303 strain in the feed was set as a treatment group, and the feed was fed to growing pigs of a total of 12 pens, four heads of growing pigs each pen, for 4 weeks. The control and treatment groups were respectively bred in independent pig pens, and to exclude the effects of the existing slurries in the pig pens, the slurries of the pig pens for the control and treatment groups were removed at the same time, before start of the experiment. From 4 days before start of the experiment, gases in the pig pens were measured with respect to the pig pens of the control and treatment groups at 9 am and 4 pm daily. Ammonia, hydrogen sulfide, complex odor, and volatile fatty acids (VOCs) were measured using a precision odor measurement device (Odor Catch, SLC-OP-1350, Scientec Lab Center Co., LTD), and methyl mercaptan was measured using a portable odorous gas monitor (MultiRAE). The daily measured data were used to calculate average values thereof for each week.

[0095] As a result, as shown in Table 3 and FIGS. 7A to 7E, the effect of reducing the odorous gases according to feeding of the CJBS303 strain was observed in all measurement items, and this effect was more pronounced at week 4 after each feeding. In particular, in the treatment group, hydrogen sulfide and methyl mercaptan which are sulfur-based odorous gases showed a reduction rate of 50% or more (Duncan, p>0.01), a complex odor showed a reduction rate of 20% or more (Duncan, p>0.05), and ammonia and volatile fatty acids showed a reduction rate of 17% and 20%, respectively.

TABLE-US-00003 TABLE 3 Complex Hydrogen Methyl odor Ammonia sulfide VOCs mercaptan (OU) (ppm) (ppm) (ppm) (ppm) Control 591.sup.a 7.7 0.88.sup.a 2.13 1.6.sup.a CJBS303 456.sup.b 6.4 0.41.sup.b 1.7 0.6.sup.b Reduction 22.80% 17.40% 53.70% 20.10% 60.30% rate (%)

[0096] EXAMPLE 4. EVALUATION OF PIG PRODUCTIVITY ACCORDING TO FEEDING OF CJBS303 STRAIN

[0097] A probiotic containing the CJBS303 strain was fed to pigs, and its effect on pig productivity was examined. Under the same conditions as in (3) of Example 3, a total of 96 heads of 74-day-old finishing pigs were divided into two groups, and a feeding experiment was performed. The average daily gain (ADG) was calculated by measuring the body weight of the individual before start of the feeding experiment and 4 week after the end of the experiment, and the average daily feed intake (ADFI) and feed conversion ratio (FCR) were calculated by measuring the daily feeding amount and balance.

[0098] As a result, as shown in Table 4, in the treatment group fed with the CJBS303 strain, ADFI was improved by 7.3% (Duncan, p<0.1), and ADG was improved by 9.0% (Duncan, p<0.03). FCR was also improved by about 0.03. These experimental results indicate that feeding of the CJBS303 strain not only reduces the amount of odorous gases, but also exhibits a positive effect on pig productivity.

TABLE-US-00004 TABLE 4 Control CJBS303 BW Start 38.69 38.66 (kg) Week 4 63.77 66.01 ADG (kg) 0.90.sup.b 0.98.sup.a ADFI (kg) 2.35.sup.b 2.52.sup.a FCR 2.62 2.59

[0099] EXAMPLE 5. VERIFICATION OF EFFECT OF REDUCING ODOROUS GASES IN PIG PEN ACCORDING TO FEEDING OF CJBS303 STRAIN

[0100] To confirm reproducibility of the effect of reducing odorous gases in a pig pen according to feeding of the CJBS303 strain, a feeding test was conducted on two pig farms. A first farmhouse (A) was selected as a growing pig pen (A-1) and a finishing pig pen (A-2), and a second farmhouse (B) was selected as a finishing pig pen, and the test subjects and the number of individuals for each farm are as in Table 5.

TABLE-US-00005 TABLE 5 A-1 A-2 B Test subject Growing pig Finishing pig Finishing pig Number of 650 heads 800 heads 944 heads individuals

[0101] Growing pigs or finishing pigs in both farms were fed with a test feed containing 2×10.sup.9 cfu/kg of the CJBS303 strain for 3 weeks, and the reduction rate was calculated by measuring the concentrations of odorous gases in the pig pens before feeding of the test feed and week 3 after feeding. Ammonia, hydrogen sulfide, and methyl mercaptan were measured using a portable odorous gas monitor (MultiRAE), and complex odor was measured using a precision odor measurement device (Odor Catch, SLC-OP-1350, Scientec Lab Center Co., LTD) at 9 am.

[0102] As a result, as shown in FIGS. 8A to 8D, in the treatment group fed with the CJBS303 strain, ammonia showed a reduction rate of 39% or more, hydrogen sulfide showed a reduction rate of 26% or more, methyl mercaptan showed a reduction rate of 24% or more, and complex odor showed a reduction rate of 21% or more. In addition, the effect of reducing the odorous gases was observed from week 3 after feeding, regardless of the growing stage or the finishing stage which are pig growth stages.

[0103] Although the above description of the present disclosure has been described for illustrative purposes, those skilled in the art will appreciate that the present disclosure may be implemented in a different specific form without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the above embodiments are not limitative, but illustrative in all aspects.

[0104] International Depositary Authority: Korean Culture Center of Microorganisms (foreign)

[0105] Accession No.: KCCM12435P

[0106] Date of deposit: 2019Feb. 13