KR102173037B1 - A novel strain of Bacillus subtilis GO and feed additive composition for improvement of meat quality of livestock comprising the same - Google Patents

Abstract

The present invention relates to a novel Bacillus subtilis GO strain (accession number: KCTC13881BP), and a feed additive composition for improving meat quality of livestock comprising the same. The novel Bacillus subtilis GO strain is isolated from traditional fermented foods, and it is confirmed that an isolated Bacillus subtilis GO strain has high stability on temperature, pH, gastric juice, bile juice, etc. In addition, by confirming that a novel Bacillus subtilis GO strain culture significantly promotes the induction of differentiation muscle cells to adipocytes as well as preadipocytes, the novel Bacillus subtilis GO strain of the present invention can be usefully used in developing feed additives or feed for improving meat quality.

Description

A novel strain of Bacillus subtilis GO and feed additive composition for improvement of meat quality of livestock comprising the same}

The present invention relates to a novel Bacillus subtilis GO strain ( Bacillus subtilis GO, accession number KCTC13881BP) and a composition for feed addition for improving meat quality of livestock comprising the same.

As dietary life becomes more advanced and diversified due to the recent increase in national income, the consumption trend of meat has shifted from quantity to quality, and the preference and sales volume for high-quality meat with good taste as well as health are increasing. In accordance with such changes in consumption, many tests and studies are being conducted to improve the quality of meat products in various ways, and in particular, studies on blended feeds or feed additives for high-quality meat production are being actively conducted.

Intramuscular fat (marbling) refers to the deposition of adipose tissue in the muscle, and is caused by intramuscular adipocytes present in the perimysium between the muscle fibers and the muscle fibers. As the number of intramuscular adipocytes increases, the marbling score improves (Kawachi, H., 2006). Improving the intramuscular fat is to maximize the taste and flavor of meat, and is a major goal of high-quality meat production. Therefore, there is a need for a technology that promotes the proliferation and differentiation of adipocytes in order to increase the intramuscular fat degree more efficiently, thereby increasing the number of adipocytes and controlling the adipose tissue to be deposited in the muscle.

It is known that the proliferation of adipocytes is not due to an increase in the number of mature adipocytes, but rather due to adipogenesis, a process of cell differentiation in which predipocytes become adipocytes.

Adipogenesis is the process of forming mature adipocytes starting from fibroblasts, converting to adipocytes, and going through very complex steps. The most striking feature of the early stage of adipogenesis is adipocytes with rounded shape of radial fibroblasts. The characteristics of the late stage are PPARγ (peroxisome proliferator-activated receptor γ), C/EBP (CCAAT/enhancer binding protein), ADD1 (adipocyte determination and differentiation-dependent). The activation of factor 1)/sterol regulatory element-binding protein 1 (SREBP1) increases the activity of enzymes involved in triglyceride synthesis and degradation, and the levels of protein and mRNA. These transcription factors are known to activate the accumulation of adipose tissue in the body through transcription of genes essential for the differentiation of adipocytes into adipocytes.

In addition, in recent studies of pigs, goats and Korean cattle, it has been reported that it is possible to convert muscle cells into fat cells. Factors that induce differentiation from muscle cells to adipocytes include external environmental factors, steroid hormones including sex hormones, and transcription factors that control the transcription of genes, all of which can be candidate factors that can improve muscle fat. .

Thus, in the process of researching feed additives that can improve muscle fat for improving meat quality of livestock, the present inventors have a novel Bacillus subtilis strain isolated from traditional fermented foods for heat, pH, gastric juice and bile juice. It was confirmed that the stability was high, and it was confirmed that intramuscular fat can be enhanced by remarkably promoting the induction of differentiation of adipocytes or muscle cells into adipocytes, thereby completing the present invention.

As a prior art, Korean Patent Publication No. 2016-0132116 describes the effect of increasing the expression level of adiponectin secreted during adipocyte differentiation of the fermented extract of Bacillus bacteria, but Bacillus bacteria is Bacillus pumilus according to the present invention. Is different from the novel Bacillus subtilis strain of. In addition, Korean Patent No. 1735759 describes an herbal probiotic in which a seed solid culture containing Bacillus subtilis for improving the meat quality of Korean cattle and an herbal preparation are mixed, but the novel Bacillus subtilis bacteria of the present invention The effect of promoting adipocyte differentiation alone is not described at all. Korea Patent Registration No. 1288392 describes a feed composition for livestock for improving meat quality of livestock, but the novel Bacillus subtilis and the effect of promoting fat differentiation of the present invention are not described or implied at all.

Korean Patent Application Publication No. 2016-0132116, Fermented extract of bacterial strains for increasing adiponectin levels, 2016. 11. 16. Publication. Registered Korean Patent No. 1735759, the effect of improving the fattening ability and meat quality of castrated Korean beef according to the supplementary benefits of herbal probiotics, 2017. 05. 08. Korean Patent Registration No. 1288392, Animal feed composition for improving meat quality and method for improving meat quality of livestock, 2013. 07. 16. Registration.

Kawachi, H., Micronutrients affecting adipogenesis in beef cattle, Animal Science Journal, 77(5), 463-471, 2006.

An object of the present invention is to provide a novel Bacillus subtilis GO strain ( Bacillus subtilis GO, accession number KCTC13881BP) and a composition for adding feed for improving the meat quality of livestock comprising the same.

The present invention relates to a novel Bacillus subtilis GO strain ( Bacillus subtilis GO, accession number KCTC13881BP).

The novel Bacillus subtilis GO strain may have adipocyte differentiation promoting activity.

In addition, the present invention relates to a composition for adding feed for improving meat quality of livestock, including the novel Bacillus subtilis GO strain culture.

The Bacillus subtilis GO strain culture can promote differentiation from adipocytes to adipocytes.

The Bacillus subtilis GO strain culture can promote differentiation from muscle cells to adipocytes.

The livestock may be one or more selected from the group consisting of cows, pigs, goats, sheep, horses, chickens, ducks and turkeys.

The present invention also, according to another aspect, relates to a feed composition for improving meat quality of livestock comprising the composition for adding the feed.

Hereinafter, the present invention will be described in detail.

The present invention relates to a novel Bacillus subtilis GO strain ( Bacillus subtilis GO, accession number KCTC13881BP).

The novel Bacillus subtilis GO strain is a strain isolated from Gochujang, has excellent protease, amylase and cellulase activity, and has excellent stability against heat, pH, gastric juice or bile juice. .

The novel Bacillus subtilis GO strain may have adipocyte differentiation promoting activity. More specifically, the novel Bacillus subtilis GO strain can produce and secrete a substance that promotes differentiation from adipocytes or muscle cells to adipocytes.

In addition, the present invention relates to a composition for adding feed for improving meat quality of livestock, including the novel Bacillus subtilis GO strain culture.

The strain culture may be a culture solution obtained by liquid culture of the novel Bacillus subtilis GO strain. The culture medium contains all of the Bacillus subtilis GO strain and the culture medium, or is a culture supernatant obtained by removing the Bacillus subtilis GO strain from the culture medium. It is preferably a culture supernatant from which Bacillus subtilis GO strain has been removed from the culture solution.

In addition, the strain culture is at least one selected from the group consisting of liquid cultured culture solution, culture solution powdered culture solution powder, and culture solution powder C1-C4 lower alcohol, acetone, n-hexane, dichloromethane, and ethyl acetate. It may be an extract extracted by adding a solvent. However, it is not limited thereto.

The culture solution powder may be obtained by powdering the culture solution obtained by liquid culture using a conventional drying method.

The C1 ~ C4 lower alcohol may be methanol, ethanol, propanol, isopropanol, butanol, and the like.

The extraction method of the extract may be used by selecting any one of methods such as hot water extraction, cold precipitation extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, compression, and the like. In addition, the desired extract may be further subjected to a conventional fractionation process, or may be purified using a conventional purification method.

In the present invention, "improving meat quality" means that intramuscular fat (marbling) is increased and the marbling score is improved, and the intramuscular fat in which adipose tissue is deposited increases. As it increases, the taste and flavor of the meat improves.

In the present invention, the "increase of intramuscular fat" is to promote the proliferation and differentiation of adipocytes to increase the number of adipocytes so that adipose tissue is deposited in the muscle. The proliferation of adipocytes means that the adipocytes become adipocytes. Can be manifested by a differentiation process that becomes It can also appear through promotion of differentiation from muscle cells to adipocytes.

The Bacillus subtilis GO strain culture may promote differentiation into adipocytes from at least one cell selected from the group consisting of adipocytes and muscle cells.

The muscle cells may be muscle cells in an undifferentiated state.

The Bacillus subtilis GO strain culture can increase the expression of adipocyte differentiation inducing factors. For example, the differentiation inducing factor may be one or more selected from the group consisting of C/EBP (CCAAT/enhancer binding protein) and PPAR (peroxisome proliferator-activated receptor γ), but is not limited thereto.

The feed-adding composition may contain 0.001 to 100% by weight of Bacillus subtilis GO strain culture based on the total weight of the feed-adding composition.

The composition for feed addition corresponds to an auxiliary feed in the feed management method.

Another aspect of the present invention relates to a feed composition for improving meat quality of livestock comprising the composition for adding feed.

The feed refers to a generally used blended feed containing appropriate nutrients to maintain the health of livestock and to fully exhibit the production capacity of the livestock, and may include all kinds of nutrient blended feed according to the type of livestock. have.

The kind of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include vegetable feeds such as grains, root fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, meals or grain by-products; Animal feeds such as proteins, inorganic logistics, oils and fats, minerals, single-cell proteins, zooplanktons or food may be used, and these may be used alone or in combination of two or more.

The content of the composition for feed addition can be adjusted within the level of those skilled in the art, and the blended feed is preferably 0.001 to 50% by weight, more preferably 0.001 to 30% by weight, and most preferably 0.001 to 10% by weight. Can be added. The content does not limit the scope of the present invention in any way.

The livestock may be one or more selected from the group consisting of cattle, pigs, goats, sheep, horses, chickens, ducks, and turkeys, but is not limited thereto.

The present invention relates to a novel Bacillus subtilis GO strain ( Bacillus subtilis GO, accession number KCTC13881BP) and a composition for feed addition for improving meat quality of livestock comprising the same, and a novel Bacillus subtilis GO strain in traditional fermented foods Was isolated, and it was confirmed that the isolated new Bacillus subtilis GO strain has high stability against temperature, pH, gastric juice and bile. In addition, it was confirmed that the novel Bacillus subtilis GO strain culture significantly promoted the induction of differentiation from muscle cells into adipocytes as well as adipocytes.

Through this, it is expected that the novel Bacillus subtilis GO strain of the present invention can be usefully used in the development of feed additives or feed for improving meat quality of livestock.

1 shows a phylogenetic analysis result using the base sequences of the 16S rRNA gene (A) and gyrB gene (B) of the GO strain, Bacillus subtilis strain isolated in the present invention.
FIG. 2 shows a result of comparing the effect of promoting adipocyte differentiation between Bacillus subtilis strains (KG20-1, GO) isolated in the present invention and the known Bacillus subtilis strains.
Figure 3 shows the results of confirming the stability against temperature of the GO strain, Bacillus subtilis strain isolated in the present invention.
FIG. 4 shows the results of confirming the stability of the GO strain, the Bacillus subtilis strain isolated in the present invention, against pH.
5 shows the results of confirming the stability of the GO strain, the Bacillus subtilis strain isolated in the present invention, against gastric juice or bile.
6 shows the results of confirming the effect of promoting differentiation from adipocytes to adipocytes according to the concentration of Bacillus subtilis GO strain culture treatment of the present invention.
7 shows the results of confirming the effect of promoting differentiation from muscle cells to adipocytes according to the concentration of Bacillus subtilis GO strain culture treatment of the present invention.
8 is a transcription factor related to adipocyte differentiation in adipocytes differentiated from adipocytes (A) or myoblasts (undifferentiated muscle cells) (B) according to the concentration of the Bacillus subtilis GO strain culture treatment of the present invention. The results of confirming the expression levels of phosphorus C/EBPα and PPARγ mRNA are shown.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the content introduced herein becomes thorough and complete, and is provided to sufficiently convey the spirit of the present invention to those skilled in the art.

< Example 1. Isolation and identification of strains>

Example 1-1. Strain isolation

To isolate the strain, samples such as soil, marine sediments, and seawater were obtained in addition to traditional fermented foods such as kimchi (cabbage kimchi, kkakdugi, goat kimchi, and dongchimi) and various types of paste (gochujang, soy sauce). The obtained sample was gradually diluted in saline, and 100 μl of the diluted sample was spread on an LB solid medium (Luria-Bertani agar plate) and cultured at 37° C. for 24 hours to isolate the strains shown in Table 1 below.

origin Strain name Kimchi KG20-1 KG20-2 soil JS50-1 JS50-2 Marine sediment Sea20-1 Sea50-1 Soy sauce Gan20-1 Gan50-1 Kochujang GO

Example 1-2. Evaluation of the enzyme activity of the strain

In order to select strains having excellent enzyme activity, protease, amylase, and cellulase activities of each strain were evaluated.

A solid medium containing the strain isolated in Example 1-1 and a substrate that specifically reacts to each enzyme was used. Specifically, the protease activity was 1% skim milk containing LB solid medium, the amylase activity 1% soluble starch containing LB solid medium, cellulase activity 1% carboxymethyl cellulose (carboxylmethyl). cellulose) containing LB solid medium was used. The strain isolated in Example 1-1 was inoculated into LB solid medium containing each substrate, and cultured at 37° C. for 24 hours. After cultivation, the transparent zones generated by the degradation of the substrate by the enzyme secreted from the strain and their size were visually observed to compare the enzyme secretion ability of each strain, and the results are shown in Table 2 below.

The enzyme secretion ability is + when it is confirmed that there is a transparent inhibitory ring around the cultured strain, the transparent inhibitory ring is clearly distinguished, but ++ when the size is small, +++ when the size of the transparent inhibitory ring is clearly observed, and a transparent inhibitory ring is observed. When it does not work, it is indicated by -.

Strain Enzyme activity Protease Amylase Cellulase KG20-1 + ++ +++ KG20-2 + + + JS50-1 + - ++ JS50-2 + - + Sea20-1 + +++ - Sea50-1 + - +++ Gan20-1 + +++ + Gan50-1 ++ - + GO ++ ++ +++

As shown in Table 2, it was confirmed that the KG20-1 and GO strains were the most excellent in all enzyme activities in protease, amylase, and cellulase.

Example 1-3. Molecular biological identification of strains

16S rRNA and gyrB gene sequencing were performed for molecular biological identification of KG20-1 and GO strains having the best enzyme activity identified in Example 1-2.

KG20-1 or GO strain was inoculated into LB liquid medium and cultured with stirring at 180 rpm at 37° C. for 15 hours to obtain a strain. From the obtained strain, gDNA (gemonic DNA) was extracted using a Genomic DNA prep kit (BIOFACT, Korea). Using the extracted gDNA as a template, 16S rRNA and gyrB genes were amplified through a polymerase chain reaction (PCR). At this time, Ex Taq polymerase (Takara, Japan) and primers (27F, 1492R, UP-1, UP-2r, UP-1S) of Table 3 were used, and PCR was performed under the PCR reaction conditions of Table 4 below. .

The 16S rRNA and gyrB genes amplified through PCR were purified using a PCR purification kit from BIOFACT. Purified 16S rRNA and gyrB genes and primers for sequencing thereof (primers for 16S rDNA gene sequencing are 27F and 1492R in Table 3 below, and primers for gyrB gene sequencing are UP- in Table 3 below. 1S, UP-2Sr, BS-F, BS-R) was used to request nucleotide sequence analysis from BIOFACT. The nucleotide sequence of each gene was compared and analyzed with the BlastN algorithm using the NCBI database.

As a result of nucleotide sequence analysis, it was confirmed that the KG20-1 and GO strains isolated in Example 1-1 were Bacillus subtilis . In addition, as can be seen from Fig. 1, which is a result of a phylogenetic comparative analysis, it was confirmed that the KG20-1 and GO strains were finally Bacillus subtilis.

Target gene Primer name Base sequence Sequence number 16S rRNA 27F 5′-AGAGTTTGATCCTGGCTCAG-3′ SEQ ID NO: 1 1492R 5′-GGTTACCTTGTTACGACTT-3′ SEQ ID NO: 2 gyrB UP-1 5′-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3′ SEQ ID NO: 3 UP-2r 5′-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3′ SEQ ID NO: 4 UP-1S 5′-GAAGTCATCATGACCGTTCTGCA-3′ SEQ ID NO: 5 UP-2Sr 5′-AGCAGGGTACGGATGTGCGAGCC-3′ SEQ ID NO: 6 BS-F 5′-GAAGGCGGNACNCAYGAAG-3′ SEQ ID NO: 7 BS-R 5′-AGCAGGGTACGGATGTGCGAGCC-3′ SEQ ID NO: 8 Y: C or T, N: A or C or G or T, R: A or G

step Reaction conditions 16S rRNA gyrB gene Pre-denaturation 94℃, 5 minutes 94℃, 5 minutes Denaturation 94℃, 2 minutes 25 cycles 94℃, 1 minute 25 cycles Annealing 55℃, 40 seconds 60℃, 1 minute Extension 72℃, 40 seconds 72℃, 2 minutes Final-extension 72℃, 10 minutes 72℃, 10 minutes

< Example 2. Adipocyte differentiation induction promoting strain screening>

As adipocytes undergo differentiation, various enzymes involved in adipogenesis, transcription factors, and hormones are expressed, resulting in an increase in the formation of lipid droplets.As a result, the formation of lipid droplets increases. Enzymes involved in the increase and fat accumulation are activated, causing fat accumulation in the cell.

Accordingly, the content of fat in adipocytes differentiated from adipocytes was measured in order to screen a strain having an effect of promoting adipocyte differentiation among the strains isolated in Example 1 above.

Specifically, in order to compare the effect of inducing adipocyte differentiation between KG20-1 and GO, Bacillus subtilis strains having excellent enzyme secretion ability among strains isolated in Example 1, and other known Bacillus subtilis strains, KCTC pre-sale 3 Species ( B. subtilis 168, KCTC3239, KCTC13112) and one of the inventors (CNF-B) were used. Each strain was inoculated in LB liquid medium and cultured for 24 hours at 37° C. and 180 rpm, and the culture solution was centrifuged to separate the supernatant, and then the separated supernatant was filtered to obtain a culture supernatant for each strain.

The adipocyte progenitor cell line 3T3-L1 cells were cultured at 37° C. in 5% CO ₂ using high glucose DMEM medium containing 10% FBS (fetal bovine serum) and 1% antibiotic/antifungal agent as a basic medium.

3T3-L1 cells, which are the adipocyte progenitor cell lines cultured above, were dispensed into a 24-well plate and cultured to fill about 80% of the plate. Afterwards, in order to induce adipocyte differentiation, use a basic medium (high glucose DMEM containing 10% FBS (fetal bovine serum) and 1% antibiotic/antifungal agent). Fat differentiation inducers (1 μM dexamethasone, 0.5 mM IBMX (3-isobuthyl-1-methylxanthine), 10 μg/ml insulin; hereinafter referred to as DM) were treated and incubated for 2 days. After that, from the 3rd day, the culture was replaced with DMEM medium containing 10% FBS and 10㎍/㎖ insulin, but the culture supernatant of each strain obtained above was added to the final 1% (v/v) for 6 days. Adipocyte differentiation was induced by culture. At this time, during the culture period of 6 days, the culture was performed by replacing with a new medium containing 10% FBS, 10 μg/ml insulin, and the culture supernatant of the strain every 2 days. Cells not treated with the culture supernatant of the strain were used as a control.

Thereafter, Oil red O staining was performed to confirm the accumulated fat in the differentiated adipocytes. Differentiation-induced adipocytes were washed with phosphate-buffered saline (PBS) and treated with 10% formalin to fix the cells. The fixed adipocytes were washed with distilled water and treated with Oil red O solution for 30 minutes at room temperature to stain the fat in the adipocytes. Oil red O solution was removed, washed three times with distilled water, and stained fat was observed through a microscope to check whether adipocytes were differentiated.To quantify the differentiated adipocytes, 100% isopropanol was added to the cells to remove the stained fat. After extraction, absorbance was measured at 520 nm. The degree of adipocyte differentiation was analyzed by converting the ratio of the absorbance of the cells treated with the culture supernatant of each strain to the absorbance of the control cells as a percentage, and the results are shown in FIG. 2.

As shown in FIG. 2, when the culture supernatant of the GO strain was treated compared to the culture supernatant of other Bacillus subtilis strains, it was confirmed that fat was accumulated in the differentiated adipocytes and the amount of fat increased.

Through this, it was found that the GO strain of the present invention has an excellent adipocyte differentiation induction promoting effect.

The new strain isolated from Gochujang, which has the effect of promoting differentiation of adipocytes, was finally named Bacillus subtilis GO, and was published in the Korean Collection for Type Cultures (KCTC) 2019. It was deposited under the accession number KCTC13881BP on July 17, 2010.

<Example 3. Stability confirmation of novel Bacillus subtilis GO strain>

The stability of the novel Bacillus subtilis GO strain having the most excellent adipocyte differentiation induction promoting effect identified in Example 2 was confirmed. Bacillus subtilis GO strain was used in the form of a freeze-dried powder.

Example 3-1. Thermal stability check

The freeze-dried powder of Bacillus subtilis GO strain was diluted in saline so that the number of bacteria became 1×10 ⁷ . After heat-treating the diluted solution at 70°C, 80°C, 90°C or 100°C for 30 minutes, it was gradually diluted in physiological saline, and 100µl of the diluted sample was spread on LB solid medium and cultured at 37°C for 24 hours to live cells. The number was confirmed. The change in the number of viable cells before (control) and after the heat treatment was confirmed using the number of viable cells, and the results are shown in FIG. 3 below.

As shown in FIG. 3, when the Bacillus subtilis GO strain was heat-treated, it was confirmed that there was little change in the number of viable cells at all of 70 to 100°C.

Through this, it was found that the novel Bacillus subtilis GO strain of the present invention has excellent stability against heat.

Example 3-2. pH stability check

Bacillus subtilis GO strain freeze-dried powder was added to 1×10 ⁷ bacteria in an LB liquid medium whose pH was adjusted to 2-9, and cultured at 25° C. for 1 hour. The culture solution was gradually diluted in physiological saline, and 100 μl of the diluted sample was plated on LB solid medium and cultured at 37° C. for 24 hours to check the number of viable cells. The change in the number of viable cells according to the pH was confirmed using the number of viable cells, and the results are shown in FIG. 4 below.

As shown in FIG. 4, when the Bacillus subtilis GO strain was exposed to various pH environments, it was confirmed that there was no change in the number of viable cells according to the pH.

Through this, it was found that the novel Bacillus subtilis GO strain of the present invention has excellent stability against pH.

Example 3-3. Stability check for gastric juice and bile juice

As for the gastric juice, a 10% pepsin (Sigma's P7125, USA) solution was filtered through a 0.2 μm syringe filter and added to the LB liquid medium so that the final concentration became 0.3% to prepare an artificial gastric juice. In addition, as a bile solution, a 10% porcine bile (Sigma's B8631) solution was filtered through a 0.2 μm syringe filter and added to the LB liquid medium so that the final concentration became 0.3% to prepare an artificial bile solution.

Bacillus subtilis GO strain freeze-dried powder was added to the artificial gastric juice or artificial bile juice so that the number of bacteria became 1×10 ⁷ and cultured at 25° C. for 1 hour. The culture solution was gradually diluted in physiological saline, and 100 μl of the diluted sample was plated on LB solid medium and cultured at 37° C. for 24 hours to check the number of viable cells. The change in the number of viable cells according to the treatment of artificial gastric juice or artificial bile fluid was confirmed using the confirmed viable count, and the results are shown in FIG. 5 below.

As shown in FIG. 5, it was confirmed that the number of viable cells when the Bacillus subtilis GO strain was treated with artificial gastric juice or artificial bile juice did not differ from the number of viable cells before treatment with artificial gastric juice or artificial bile juice (control).

Through this, it was found that the novel Bacillus subtilis GO strain of the present invention has excellent stability against gastric juice or bile juice.

<Example 4. Confirmation of the effect of promoting adipocyte differentiation induction of novel Bacillus subtilis GO strain>

Example 4-1. Confirmation of the effect of promoting differentiation from adipocytes to adipocytes of the culture supernatant of Bacillus subtilis GO strain

In order to confirm the specific adipocyte differentiation induction promoting effect of the novel Bacillus subtilis GO strain in which the effect of promoting differentiation from adipocytes to adipocytes of Example 2 was confirmed, the same method as in the induction of adipocyte differentiation of Example 2 Adipocyte differentiation was induced from adipocytes. At this time, in the experimental group, cells not treated with anything (untreated group), cells not treated with Bacillus subtilis GO strain culture supernatant (control group), and Bacillus subtilis GO strain culture supernatant at a final concentration of 1%, 2% or It proceeded by dividing into the treated cells to be 3%. The degree of adipocyte differentiation was analyzed by converting the absorbance of the cells treated with the Bacillus subtilis GO strain culture supernatant or the cells of the untreated group as a percentage to the absorbance of cells not treated with the control Bacillus subtilis GO strain culture supernatant. , The results are shown in FIG. 6.

As shown in Figure 6, compared to the control group, when the Bacillus subtilis GO strain culture supernatant was treated, the amount of fat increased, and it was confirmed that the amount of fat increased in a concentration-dependent manner with the Bacillus subtilis GO strain culture supernatant.

Through this, it was found that the novel Bacillus subtilis GO strain of the present invention exhibits excellent adipocyte differentiation induction promoting effect by secreting an effective substance for adipocyte differentiation induction.

Example 4-2. Confirmation of the effect of promoting differentiation from muscle cells to adipocytes of the culture supernatant of Bacillus subtilis GO strain

In order to confirm that the effect of promoting adipocyte differentiation of the novel Bacillus subtilis GO strain of the present invention promotes the induction of differentiation from muscle cells to adipocytes, after inducing differentiation into adipocytes using muscle cells, differentiated fat The amount of fat in the cells was analyzed.

C2C12 cells, which are myoblasts (muscle cells in an undifferentiated state), use high glucose DMEM medium containing 10% FBS (fetal bovine serum) and 1% antibiotics/antifungal agents as the basic medium, under conditions of 5% CO ₂ . Incubated at 37°C.

The myoblasts cultured above were used to induce differentiation from myoblasts to adipocytes in the same manner as in the induction of adipocyte differentiation in Example 2. At this time, in the experimental group, cells not treated with anything (untreated group), cells not treated with Bacillus subtilis GO strain culture supernatant (control group), and Bacillus subtilis GO strain culture supernatant at a final concentration of 1%, 2% or It proceeded by dividing into the treated cells to be 3%. The degree of adipocyte differentiation was analyzed by converting the absorbance of cells treated with the culture supernatant of Bacillus subtilis GO strain as a control or the cells treated with the culture supernatant of Bacillus subtilis GO strain as a percentage to the non-treated cells. And the results are shown in FIG. 7.

As shown in FIG. 7, when the Bacillus subtilis GO strain culture supernatant was treated as compared to the control, the amount of fat increased, and the amount of fat increased depending on the concentration of the Bacillus subtilis GO strain culture supernatant.

Through this, it was found that the novel Bacillus subtilis GO strain culture of the present invention promotes the induction of differentiation into adipocytes from muscle cells as well as adipocytes.

Example 4-3. Confirmation of gene expression related to inducing differentiation into adipocytes

In order to confirm whether the novel Bacillus subtilis GO strain of the present invention influences the increase in mRNA expression of adipocyte differentiation-related genes in promoting the induction of differentiation into adipocytes, the mRNA of the adipocyte differentiation-related transcription factors C/EBPα and PPARγ The expression level was analyzed.

Differentiation into adipocytes was induced from adipocytes (3T3-L1) or non-differentiated myoblasts (C2C12) as in Examples 4-1 and 4-2, and differentiation-induced adipocytes were collected and total Total RNA was isolated using an RNA prep kit (BIOFACT). The isolated total RNA was used as a template, and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (TOYOBO Co., Japan). Using the synthesized cDNA as a template, real time PCR (real time PCR) was performed using SYBR Green Supermix (Bio-radt, USA) and the primers in Table 5 below to compare the mRNA expression levels of C/EBPa and PPARγ, The results are shown in FIG. 8. The mRNA expression level of β-actin was used as a quantitative control.

Target gene Primer name Base sequence Sequence number C/EBPα C/EBP-S 5′-AGABATCAGCGCCTACATCG-3′ SEQ ID NO: 9 C/EBP-AS 5′-TGTAGGTGCATGGTGGTCTG-3′ SEQ ID NO: 10 PPARγ PPAR-S 5′-ATGGAAGACCACTCGCATT-3′ SEQ ID NO: 11 PPAR-AS 5′-CATGGACACCATACTTGAG-3′ SEQ ID NO: 12 β-actin Actin-S 5′-CCACAGCTGAGAGGGAAATC-3′ SEQ ID NO: 13 Actin-AS 5′-AAGGAAGGCTGGAAAAGAGC-3′ SEQ ID NO: 14 B: C or G or T

As shown in FIG. 8, it was confirmed that the mRNA expression levels of C/EBPα and PPARγ in adipocytes differentiated from adipocytes (A) and myoblasts (B) increased in a concentration-dependent manner in the culture supernatant of Bacillus subtilis GO strain. I did.

Through this, the novel Bacillus subtilis GO strain culture of the present invention promotes the induction of differentiation into adipocytes by increasing the expression of the transcription factors C/EBPα and PPARγ related to adipocyte differentiation in both adipocytes and muscle cells. I could see that.

<110> VITA BIO CO., LTD <120> novel strain of Bacillus subtilis GO and feed additive composition for improvement of meat quality of livestock comprising the same <130> 19-0027 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R <400> 2 ggttaccttg ttacgactt 19 <210> 3 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> UP-1 <400> 3 gaagtcatca tgaccgttct gcaygcnggn ggnaarttyg a 41 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> UP-2r <400> 4 agcagggtac ggatgtgcga gccrtcnacr tcngcrtcng tcat 44 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> UP-1S <400> 5 gaagtcatca tgaccgttct gca 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> UP-2Sr <400> 6 agcagggtac ggatgtgcga gcc 23 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> BS-F <400> 7 gaaggcggna cncaygaag 19 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> BS-R <400> 8 agcagggtac ggatgtgcga gcc 23 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C/EBP-S <400> 9 agabatcagc gcctacatcg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> C/EBP-AS <400> 10 tgtaggtgca tggtggtctg 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PPAR-S <400> 11 atggaagacc actcgcatt 19 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> PPAR-AS <400> 12 catggacacc atacttgag 19 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Actin-S <400> 13 ccacagctga gagggaaatc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Actin-AS <400> 14 aaggaaggct ggaaaagagc 20

Claims (7)

A novel Bacillus subtilis GO strain (Bacillus subtilis GO, accession number KCTC13881BP). The method of claim 1,
The novel Bacillus subtilis GO strain is Bacillus subtilis GO strain, characterized in that it has adipocyte differentiation promoting activity. A composition for promoting adipocyte differentiation, characterized in that it comprises the novel Bacillus subtilis GO strain culture of claim 1. The method of claim 3,
The Bacillus subtilis GO strain culture composition for promoting adipocyte differentiation induction, characterized in that to promote the differentiation of adipocytes from adipocytes. The method of claim 3,
The Bacillus subtilis GO strain culture is a composition for promoting adipocyte differentiation induction, characterized in that promoting differentiation from muscle cells to adipocytes. A composition for adding livestock feed comprising the composition for promoting adipocyte differentiation induction according to any one of claims 3 to 5. The method of claim 6,
The cattle are cattle, pigs, goats, sheep, horses, chickens, ducks and turkey composition for adding livestock feed, characterized in that at least one selected from the group consisting of.

Images