KR101357118B1 - Probiotics with improved stability using jujube extracts - Google Patents

Abstract

The present invention relates to a probiotic with increased stability containing a new Bacillus coagulans KBC-KC1 strain cultured using jujube extract as an active ingredient, more specifically jujube extract No. 1 Bacillus coagulase Cabici-Cassie 1 strain cultured in the medium containing the increased spore-forming ability, heat resistance and storage stability, probiotics, feed additives, pharmaceutical composition for immune enhancement and lactic acid bacteria It can be usefully used as a formulation.

Description

Probiotics with improved stability using jujube extracts}

The present invention relates to improving the stability by promoting the sporulation of Bacillus coagulans, which are widely used as feed probiotics for animal activity and immune improvement by using jujube extract as a culture medium.

Jujube is a deciduous deciduous tree belonging to the sea buckthorn family, and there are two species with different ecotypes, such as Chinese jujube and Indian. The traditional varieties produced in Korea are 'boeun jujube' harvested from Boeun, Chungcheongbuk-do for a long time, and 'democulture' grown in Gyeongsang Province, and due to the nature of fruit, they cannot belong to the jujube superior varieties. There is a variety called 'Sanjo' which is used as a medicine or as a herbal medicine. Jujube is an alkaline food rich in vitamin C and contains a large amount of minerals such as calcium, phosphorus, and iron, which are known to reduce heat, relieve constipation, and stop coughing. It is also effective in stiffening, anti-aging, and nerve relieving effects. Recently, intestinal harmful enzyme inhibitory effect and antioxidant effect have been revealed.

Since its discovery in 1928, antibiotics have been used extensively in livestock specifications for the treatment and growth of livestock diseases since the 1950s. At the sub-therapeutic level, antibiotics were mixed with livestock feed and used in livestock farming to improve productivity, including disease control, growth, and feed efficiency, and contributed to income for livestock farmers. In recent years, however, the trend has been gradually regulated due to the emergence of resistant strains due to misuse of antibiotics and the safety problems caused by residual antibiotics in livestock products. Since 2006, the EU has banned the use of antibiotics, which are used to promote livestock growth. In addition to the changes in the livestock environment, the need for development of antibiotic substitutes is increasing as consumers' interest in safe livestock products and the demand for livestock without antibiotics are increasing.

Antibiotic substitutes include probiotics, acidifers, immunomodulatory and enhancers, feed enzyme complexes, and functional natural extracts, but probiotics that have beneficial effects on host animals by improving intestinal microbial balance. Is most in the limelight. Probiotics are generally defined as live microbial food ingredients that are beneficial to health (Liilly and Stillwell, Science 147: 747-748, 1965), but recently Salminen et al . ( Int . J. Food) . Microbiol . 44: 93-106, 1998) are defined as living microbial products in food and feed or dietary additives designed to promote human or animal health. Probiotic Lactobacillus is most effective when it is able to settle in the intestine to proliferate and inhabit. Settling to the intestinal cell wall is an important prerequisite for intestinal colonization. It is known that probiotic lactic acid bacteria can have various beneficial effects in the intestine by fixing on the intestinal cell surface (Donohue et. al , Asia pacific J. Clin . Nutr . 5: 25-28, 1996). Settlement demonstrates the potential for probiotic lactic acid bacteria to enter the intestine and for lactic acid bacteria to grow in intestinal conditions. In addition, lactic acid bacteria must be resistant to oral and stomach enzymes, low pH of gastric juice, bile and mucus in the small intestine in order to function well in the intestine (Havenaar et. al ., Probiotics : the Scientific Basis (Fuller, R., ed.), Pp. 209-224, 1992). In addition, these probiotics settle in the intestines of livestock in the livestock sector, inhibiting the growth of other harmful microorganisms, and aid in the digestion and absorption of the feed consumed, thereby promoting the growth of livestock and improving feed efficiency, thus improving the productivity of ruminants. It is important to select and utilize probiotics that are suitable for this purpose.

Bacillus coagulans , known as diffuse spores, is a Gram-positive bacillus with motility. It is stable because it forms spores and does not die in the intestine when added to feeds. It is known to function normally by normalizing the bacterial flora and is widely used as feed probiotic for livestock farms. However, microorganisms used in industry form spores that are relatively resistant to the removal of other environmental factors under the conditions of fermentation tanks. Therefore, even though spores are formed, the concentration of effective microorganisms rapidly increases after a long storage and distribution period. Fall off.

One of the reasons why the effectiveness of probiotics developed and marketed in Korea has been evaluated as insignificant is that it is impossible to add probiotics with an appropriate number of effective bacteria to the blended feed, and thus, the expected effects of probiotics cannot be achieved. One of the causes of the lack of effective bacteria of such probiotics can be said to be due to the production process of probiotics. No matter how high the concentration of the probiotic culture, most of the culture medium takes up the moisture to be removed by drying, and in order to maintain the activity of the probiotic, it is mainly used a method such as lyophilization or spray drying. Since expensive drying facilities are needed, the drying cost is a large part of the cost of producing probiotics. Therefore, if probiotics are developed by forming spores resistant to environmental stresses in an efficient way to lower production costs, it is possible to produce products in which the concentration of effective microorganisms is maintained even after long-term storage.

Thus, the inventors of the present invention are cultured in a medium containing jujube extract Bacillus coagulase Cavy-Cassie 1 ( Bacillus The present invention was completed by confirming that the spore forming ability of the coagulans KBC-KC1) strain was greatly improved, and that the spores had thermal stability and storage stability.

It is an object of the present invention to provide a novel Bacillus coagulans KBC-KC1 strain.

In addition, another object of the present invention Bacillus coagulase Cabisi-Cassie 1 ( Bacillus 1) in a medium containing jujube extract coagulans KBC-KC1) to provide a method for culturing the strain.

In addition, another object of the present invention is a novel Bacillus coagulase Cabisi-Cassie 1 ( Bacillus coagulans KBC-KC1) strain, a culture of the strain or to provide a probiotics composition comprising a spore formed from the strain.

In addition, another object of the present invention Bacillus coagulase Cabici-Cassie 1 ( Bacillus) cultured in a medium containing jujube extract coagulans KBC-KC1) strain, culture of the strain or to provide a method for producing a probiotic comprising a spore formed from the strain.

In addition, another object of the present invention is a Bacillus coagulans KBC-KC1 strain ( Bacillus coagulans KBC-KC1) strain, a culture solution of the strain or spores formed from the strain, or a feed additive containing a probiotic composition as an active ingredient To provide.

In addition, another object of the present invention is Bacillus coagulans KBC-KC1 ( Bacillus coagulans KBC-KC1) strain, the culture of the strain or spores formed from the strain, or the immunity of livestock containing the probiotic composition as an active ingredient It is to provide a pharmaceutical composition for enhancement.

In addition, another object of the present invention is to provide a Bacillus coagulans KBC-KC1 strain ( Bacillus coagulans KBC-KC1) strain, a culture supernatant thereof, or a probiotic composition containing a probiotic composition as an active ingredient.

In order to achieve the above object, the present invention is deposited Bacillus coagulase Cavy-Cassie 1 ( Bacillus) deposited with accession number KACC91677P coagulans KBC-KC1) strain.

In addition, the present invention comprises culturing the strain in a medium containing a jujube extract Bacillus coagulase Cavy-Cassie 1 ( Bacillus It provides a method for culturing coagulans KBC-KC1) strain.

The present invention also provides a novel Bacillus coagulans KBC-KC1 strain, a culture of the strain or a probiotics composition comprising spores formed from the strain.

In addition,

1) culturing the strain of claim 1 in a medium containing the jujube extract; And

2) obtaining the spores formed from the cultures of the strain, the culture of the strain or the strain from the culture cultured in step 1), Bacillus coagulase Cabici-Cassie 1 ( Bacillus coagulans KBC-KC1) provides a method for producing a probiotic from the strain.

The present invention also provides a Bacillus coagulans KBC-KC1 strain ( Bacillus coagulans KBC-KC1) strain, a culture medium of the strain or spores formed from the strain, or a feed additive containing the probiotic composition as an active ingredient.

In addition, the present invention is Bacillus coagulans KBC-KC1 strain ( Bacillus coagulans KBC-KC1) strain, the culture of the strain or spores formed from the strain, or a pharmaceutical composition for immune enhancement of livestock containing the probiotic composition as an active ingredient To provide a composition.

In addition, the present invention provides a Bacillus coagulans KBC-KC1 strain ( Bacillus coagulans KBC-KC1) strain, a culture of the strain or spores formed from the strain, or a lactic acid bacteria preparation containing the probiotic composition as an active ingredient.

Bacillus coagulans KBC-KC1 strain of the present invention, when cultured in a culture medium to which hot water or ethanol jujube extract is added, the sporulation ability is greatly increased, Since the storage stability is improved, it can be usefully used as probiotics, feed additives, immune enhancing pharmaceutical compositions, and lactic acid bacteria preparations.

Figure 1 shows the results of homology analysis of the 16S rRNA gene sequence obtained by using a blast (blast).
FIG. 2 shows spores of Bacillus coagulitis Cabici-Case 1 as observed by scanning electron microscopy. FIG.
Figure 3 is a feeder cell of Bacillus coagulase Cabici-Cassie 1 observed by scanning electron microscopy.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel Bacillus coagulans KBC-KC1 strain.

The strain preferably has a 16S rRNA nucleotide sequence as set forth in SEQ ID NO: 1, more preferably deposited with accession number KACC91677P, but is not limited thereto.

The Bacillus coagulase Cabici-Cashi 1 is preferably derived from kimchi, but is not limited thereto.

In addition, the present invention includes the step of culturing Bacillus coagulase Cabici-Cassie 1 ( Bacillus) comprising culturing in a medium containing jujube extract It provides a method for culturing coagulans KBC-KC1) strain.

The jujube extract,

1) extracting by adding an extraction solvent to the jujube;

2) filtering the extract of step 1); And

3) The filtered extract of step 2) may be prepared by a method comprising the steps of concentration under reduced pressure and drying to prepare a jujube extract, but is not limited thereto.

The jujube extract is preferably added in 1 to 10% by weight relative to the total weight of the medium, but is not limited thereto.

In the above method, the jujube of step 1) can be used without limitation, such as grown or commercially available. The jujube may use the whole jujube, the seed may be used, but is not limited thereto.

The extraction solvent is preferably water, alcohol or a mixture thereof. The alcohol is C ₁ to C ₂ It is preferable to use a lower alcohol, it is preferable to use ethanol or methanol as the lower alcohol, more preferably using 70% ethanol, but is not limited thereto. As extraction methods, conventional methods in the art, such as filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction, can be used, and the extraction is preferably performed once to five times by hot water extraction, and three times to extract repeatedly. More preferably, but is not limited thereto. The extraction solvent may be added 1 to 20 times the weight of the crushed dry jujube or raw jujube, preferably 5 to 15 times. Extraction temperature is preferably 80 to 120 ℃ but is not limited thereto. In addition, the extraction time is preferably 1 to 13 hours, but is not limited thereto.

In the above method, the vacuum concentration of step 3) is preferably a vacuum vacuum concentrator or a rotary vacuum concentrator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

Bacillus coagulase Cabisi-Cassie 1 ( Bacillus coagulans KBC-KC1) strain is preferably cultured in nutrient medium or MRS medium for 3 to 7 days at 20 to 50 ℃, shaking culture in aerobic conditions, but is not limited to this, usually Cultivation is possible by culturing bacteria.

In a specific embodiment of the present invention, the present inventors sampled from the kimchi and selected strains using a medium for lactic acid bacterium separation. As a result of analyzing the homology of the 16S rRNA gene sequence (SEQ ID NO: 1) obtained after culturing the selected strain, 99% of the standard strain sequence of Bacillus coagulans coincides with the selected strain. Bacillus 1 coagulans KBC-KC1) '(see FIG. 1), and the strain was deposited on September 21, 2011 to the Agricultural Genetic Resource Information Center (deposit number; KACC91677P).

In addition, the inventors of the present invention Bacillus coagulase Cabici-Cassie 1 ( Bacillus 1) selected in the nutrient agar medium containing jujube extract in a standardized content of 1 to 10% Coagulans KBC-KC1) strains were inoculated and cultured to determine the spore forming ability of the strain, Bacillus coagulans Cabici-Cassie 1 ( Bacillus coagulans KBC-KC1) strains, the standard strains of Bacillus core Tangerine Lawrence (Bacillus coagulans KACC 10117) showed higher sporulation ability, and when cultured in a medium containing jujube extract, the spore forming ability was significantly increased depending on the concentration. In particular, in a medium containing 10% of hydrothermal jujube extract, It was confirmed that the sporulation ability increased by 5 times (see Table 1).

Through this, the present invention provides a novel Bacillus coagulans KBC-KC1 strain (Accession No. KACC91677P) having superior spore forming ability, heat resistance, and storage stability in a medium containing jujube extract. Prepared.

In addition, the inventors of the present invention, Bacillus coagulose Cabici-Cassie 1 ( Bacillus 1) screened on the nutrient agar medium containing standardized jujube extract Inoculation and culture of the coagulans KBC-KC1) strain confirmed the spore formation ability of the strain, and Bacillus coagulase Cabici-Cassie 1 ( Bacillus) cultured in a medium containing jujube extract coagulans KBC-KC1) strains, the standard strains of Bacillus core Tangerine Lawrence (Bacillus coagulans KACC 10117) showed significantly higher sporulation ability (see Table 1) and higher heat resistance (see Table 2). In addition, the Bacillus coagulus Cabici-Cassie 1 strain in the general nutrient medium has a higher storage stability than the standard strain, and spores of the Bacillus coagulus Cabici-Cassie 1 strain were cultured in the jujube extract containing medium spores of the standard strain It was confirmed that the storage stability is significantly higher than that (see Table 3).

In addition, the present invention is Bacillus coagulans KBC-KC1 ( Bacillus coagulans KBC-KC1) strain (Accession No. KACC91677P), the culture of the strain or It provides a probiotics composition comprising spores formed from the strain.

In addition,

1) culturing the strain of claim 1 in a medium containing the jujube extract; And

2) the strain from the culture medium cultured in step 1), the culture solution of the strain or Obtaining spores formed from the strain, Bacillus coagulase Cabici-Cassie 1 ( Bacillus coagulans KBC-KC1) provides a method for producing a probiotic from the strain.

The jujube extract is characterized by being extracted using water, C ₁ to C ₂ lower alcohol or a mixture thereof as a solvent, but is not limited thereto.

Bacillus coagulase Cabisi-Cassie 1 ( Bacillus coagulans KBC-KC1) strain is preferably cultured in nutrient medium or MRS medium for 3 to 7 days at 20 to 50 ℃, shaking culture under aerobic conditions, but is not limited thereto, usually Cultivation is possible by culturing bacteria.

Therefore, when Bacillus coagulase Cabici-Cassie 1 strain of the present invention is cultured in a medium containing jujube extract, it forms more spores than the standard strain, has high heat resistance, high storage stability, Bacillus coagulase Cabisi -Casey 1 ( Bacillus coagulans KBC-KC1) strain (Accession No. KACC91677P), culture of the strain or Spores formed from the strain can be used for animals, including livestock as a probiotics composition, can be added to food or animal feed, it can be usefully used as animal medicines.

Bacillus coagulase Cabisi-Cassie 1 of the present invention ( Bacillus coagulans KBC-KC1) strain (Accession number KACC91677P), a culture solution of the strain or Probiotics comprising spores formed from the strains can be administered orally or parenterally during clinical administration, and 1 strain of Bacillus coagulase Cabici-Cassie, the main component of the strain, the culture medium of the strain, or an effective amount of spores formed from the strains. Species or two or more pharmaceutically acceptable conventional carriers or one or two or more additives may be selected to prepare the compositions in conventional formulations.

The carrier may be selected from one or more selected from among diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives. Examples of the additives include one or more of flavorings, vitamins and antioxidants Can be used.

In the present invention, the carrier and the additive may be used in any pharmaceutically acceptable, specifically, as a diluent lactose (lactose monohydrate), corn starch (cornstarch), soybean oil (soybean oil), microcrystalline cellulose (microcrystalline cellulose) ) Or mannitol (D-mannitorl) is preferable, magnesium stearate or talc is preferred as a lubricant, polyvinyipyrolidone (PVP: polyvinyipyrolidone) or hydroxypropyl cellulose (HPC :) hydroxypropylcellulose). In addition, the disintegrant may be selected from among carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polyacrylin potassium or cross-linked polyvinylpyrrolidone. Preferably, the sweetener is selected from sucrose, fructose, sorbitol or aspartame, and the stabilizer is carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin, It is selected from white bee's wax or xanthan gum, and preservatives include methyl p-hydroxy benzoate (methhlparaben), propyl p-hydroxy benzoate (propylparaben), or sorbic acid. It is preferable to select from potassium sorbate.

In addition, the present invention provides a feed additive containing Bacillus coagulans KBC-KC1 strain, a culture of the strain or spores formed from the strain, or a probiotic composition comprising the same as an active ingredient. do.

The feed additives may be prepared in the form of additives for feed for livestock and fish.

The feed additive is a Bacillus coagulans KBC-KC1 strain, a culture solution of the strain or spores formed from the strain, or 1 to 100 parts by weight of a probiotic composition comprising the same. It is preferred to add 20% by weight, more preferably 10 to 20 parts by weight, but is not limited thereto.

The feed additive includes the Bacillus coagulans KBC-KC1 strain, a culture solution of the strain or spores formed from the strain, or a probiotic composition comprising the same, from 0.1 to 20% by weight, lipolysis. 0.001 to 0.01% by weight of enzyme, 1 to 20% by weight of tricalcium phosphate, 0.01 to 0.1% by weight of vitamin E, 1 to 10% by weight of enzyme powder, 0.1 to 10% of lactic acid bacteria Part by weight and glucose is preferably composed of 20 to 90% by weight, but is not particularly limited thereto, Bacillus coagulans KBC-KC1 strain, culture of the strain or from the strain If the spores formed or the probiotic composition containing the same are added in an effective amount, they can be used as the feed additive of the present invention.

The feed additive may additionally contain a carrier such as grains and by-products that are acceptable to poultry and livestock. In the present invention, the feed additive may be added as it is or a known carrier, stabilizer and the like may be added. Various nutrients such as vitamins, amino acids and minerals, antioxidants, antibiotics, antibacterial agents and other additives may be added And the shape thereof may be a suitable state such as powder, granule, pellet, suspension and the like. When the feed additive of the present invention is supplied, it can be supplied to poultry, livestock and the like singly or mixed with feed.

In addition, the present invention is a Bacillus coagulans KBC-KC1 strain ( Bacillus coagulans KBC-KC1) strain, the culture of the strain or spores formed from the strain, or enhance the immunity of livestock containing a probiotic composition comprising the same as an active ingredient It provides a pharmaceutical composition.

In addition, the present invention provides a Bacillus coagulans KBC-KC1 strain ( Bacillus coagulans KBC-KC1) strain, a culture solution of the strain or spores formed from the strain, or a lactic acid bacteria preparation containing a probiotic composition comprising the same as an active ingredient. do.

Hereinafter, the present invention will be described in detail by Examples, Experimental Examples, and Formulation Examples.

However, the following Examples, Experimental Examples, and Formulation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Formulation Examples.

< Example  1> Isolation and Screening of New Microorganisms from Kimchi Samples

The present inventors isolated the lactic acid bacteria from kimchi to select a new strain.

Specifically, samples from the kimchi produced in Jinju area were diluted with 0.9% NaCl, inoculated in MRS (De Man, Rogosa, Sharpe) agar medium (Difco, USA), a medium for lactic acid bacterium separation, 37 Strains were selected by incubation at 42 ° C. for 42 hours. Then, for species analysis of the selected strains, microorganisms were sought for macrogens, cultured the strains, genomic DNA was extracted, and the nucleotide sequence of 16s rDNA 1515bp was determined (SEQ ID NO: 1). As a result of analyzing the homology of the sequencing of the species analysis result of the identified blast (blast), the selected strain was found to be a species belonging to the genus Bacillus (Fig. 1). The selected strain is Bacillus core tangerine lance (Bacillus coagulans) and a 99% match to the selected strain showed similarity "Bacillus core tangerine lance KB upon which of these - Casey 1 (Bacillus coagulans KBC-KC1) ", which was deposited on September 21, 2011 to the Genetic Resources Information Center (Accession Number; KACC91677P).

< Example  2> Preparation of Jujube Extract

<2-1> Heat number  Preparation of Jujube Extract

The present inventors prepared hot water jujube extract to add to the medium of the strain selected in <Example 1>.

Specifically, 1 L of water was added to 100 g of crushed dried jujube or raw jujube, hot water extracted and filtered with a shaker at 110 ° C. for 5 hours to obtain jujube extract and jujube seed extract, and then concentrated under reduced pressure with a rotary vacuum concentrator. In addition, hydrothermal jujube extract was standardized by the manufacturing method of Korea Pharmacopoeia Liquid Extract (about 20% solids).

<2-2> Preparation of Ethanol Jujube Extract

The present inventors prepared the ethanol jujube extract to add to the medium of the strain.

Specifically, 1 g of ethanol (70% v / v) was added to 100 g of ground dried jujube or raw jujube, and then suspended at room temperature for 8 hours, and filtered through a 0.45 μm filter (Millipore Co, USA) to obtain an ethanol fraction. It concentrated under reduced pressure with a rotary vacuum concentrator. At this time, the ethanol jujube extract was standardized by the manufacturing method of Korea Pharmacopoeia liquid extract (solid about 4%).

< Experimental Example  1> Bacillus Coagulant Kabysi - Casey  One( Bacillus coagulans KBC - KC1 Spores of Formability  Confirm

The present inventors confirmed the spore-forming ability to determine the effect of jujube extract on the spore-forming ability of Bacillus coaculance Cabici-Case 1.

Specifically, Bacillus coagulance Cavy-Cassie 1 ( Bacillus coagulans KBC-KC1) and standard strains of Bacillus core Tangerine Lance (Bacillus coagulans KACC 10117 (preparation of Agricultural Genetic Resource Information Center) was incubated in a nutrient medium (Nutrient broth, Difco, USA) for 24 hours at 37 ° C., and the initial bacterial count was measured and used as a control. After inoculating 100 μl of Bacillus coagulans Cabisi-Cassie 1 and Bacillus coagulans bacteria in nutrient agar medium containing 1, 5 and 10% by weight of extract or 70% ethanol jujube extract, incubated at 37 ° C. for one week. To produce spores. Spores of the two strains produced were observed by crystal violet staining with an optical microscope (Olympus, Model: SZ-ST, Japan). Spores (FIG. 2) and vegetative cells (FIG. 3) were observed using a scanning electron microscope (Carl Zeiss LEO. 1420 scanning electron microscope, Germany) to confirm the microstructural characteristics of the bacteria. In order to measure the number of spores, 5 ml of sterile distilled water was added to each agar medium, and the collected spores evenly scraped from the spores grown on the plate surface with a sterile swab were divided into 0.1 ml portions of the eppendorf tube. After 15 minutes of treatment in a shaker (Vision Science, Korea) at 80 ℃ and germinated, the germinated bacteria were incubated for 24 hours at 37 ℃ by serial dilution using a nutrient medium to measure the concentration of spores.

As a result, the Bacillus coagulase Cabici-Case 1 strain showed a higher spore-forming ability than the standard strain, it was confirmed that the concentration of the hot water extracted jujube extract or ethanol jujube extract (Table 1).

Determination of Spore Formation of Bacillus Coagulitis Cabicy-Case 1 Medium composition Spore Population (cfu / ml) B. coagulans KACC 10117 B. coagulans KBC-KC1 Nutrient Agar 1.8 x 10 ⁴ 1.9 × 10 ⁴ Nutritional Medium + Jujube Hot Water Extract 1% 2.1 x 10 ⁴ 3.3 × 10 ⁶ Nutritional Medium + Jujube Hydrothermal Extract 5% 2.0 x 10 ⁴ 6.1 × 10 ⁶ Nutritional Medium + Jujube Hot Water Extract 10% 1.9 × 10 ⁴ 9.4 × 10 ⁶ Nutritional Medium + Jujube Ethanol Extract 1% 2.2 x 10 ⁴ 2.0 × 10 ⁶ Nutritional Medium + Jujube Ethanol Extract 5% 2.3 x 10 ⁴ 4.1 × 10 ⁶ Nutritional Medium + Jujube Ethanol Extract 10% 1.8 x 10 ⁴ 6.8 × 10 ⁶

< Experimental Example  2> Bacillus Coagulant Kabysi - Casey  One( Bacillus coagulans KBC - KC1 ) Check the heat resistance of the spores

The present inventors confirmed the heat resistance of the spores in order to determine the effect of jujube extract on the heat resistance of Bacillus Coaculance Cabici-Cassie 1 spores.

Specifically, Bacillus coagulance Cavy-Cassie 1 ( Bacillus coagulans KBC-KC1) and standard strains of Bacillus core Tangerine Lance (Bacillus coagulans KACC 10117 (preparation of Agricultural Genetic Resource Information Center) was incubated in a nutrient medium (Nutrient broth, Difco, USA) for 24 hours at 37 ° C., and the initial bacterial count was measured and used as a control. Inoculate 100 μl of the Bacillus coagulans Cabisi-Cassie 1 and Bacillus coagulans bacteria in a nutritious agar medium containing 10% by weight of the extract or ethanol jujube extract, and incubate for one week at 37 ° C. to produce spores. It was. To measure the number of spores generated, add 5 ml of sterile distilled water to each agar medium, collect and scrape spores grown on the plate surface with a sterile swab, and divide into 0.25 ml Eppendorf tubes. The number of bacteria measured by incubation at 90 ℃ shaking culture (Vision Science, Korea) up to 21 minutes at 3 minutes intervals, and then cooled in ice water immediately after heat treatment and incubated for 24 hours at 37 ℃ using a nutrient agar medium. D-value was obtained through.

As a result, when the Bacillus coagulitis Cabici-Cassie 1 was incubated in a nutrient medium containing hot water extract or ethanol extract, the D _{90 ℃} -value was 9.8 minutes or 8.9 minutes. It confirmed that it showed higher heat resistance (Table 2).

Thermal Stability of Bacillus Coagulans Cabicy-Cassie Spores Medium composition D-value (minutes) ¹ B. coagulans KACC 10117 B. coagulans KBC-KC1 Nutrition badge 5.1 ± 0.47 5.8 ± 0.34 Nutritional Medium + Jujube Hot Water Extract 10% 5.5 ± 0.22 9.8 ± 0.28 Nutritional Medium + Jujube Ethanol Extract 10% 5.3 ± 0.32 8.9 ± 0.13

¹ Measured data is ± standard deviation by 3 repetitions

< Experimental Example  3> Bacillus Coagulant Kabysi - Casey  One( Bacillus coagulans KBC - KC1 ) Storage stability of spores

The present inventors confirmed the effect of jujube extract on the storage stability of Bacillus coaculans Cabici-Cassie 1 spores.

Specifically, Bacillus coagulance Cavy-Cassie 1 ( Bacillus coagulans KBC-KC1) and standard strains of Bacillus core Tangerine Lance (Bacillus coagulans KACC 10117 (preparation of Agricultural Genetic Resource Information Center) was incubated in a nutrient medium (Nutrient broth, Difco, USA) for 24 hours at 37 ° C., and the initial bacterial count was measured and used as a control. Inoculate 100 μl of the Bacillus coagulans Cabisi-Cassie 1 and Bacillus coagulans bacteria in a nutritious agar medium containing 10% by weight of the extract or ethanol jujube extract, and incubate for one week at 37 ° C. to produce spores. It was. 5 ml of sterile distilled water was added to each agar medium in which spores were produced, and evenly collected by scraping spores grown on the surface of the plate with a sterile cotton swab, divided into 0.25 ml eppendorf tubes, and stored at room temperature. And preserved for 6 months. Thereafter, the germination rate of the spores was determined by counting the number of spores through the measured bacterial counts by culturing for 24 hours at 37 ° C. using a nutrient agar medium to examine the storage stability of the spores.

As a result, spores decreased as time passed, but spore germination rate with time-dependent change in Bacillus coagulant Cabici-Cassie 1 was cultured using hot water extract and ethanol extract. It was confirmed that significantly higher than (Table 3).

Storage Stability of Bacillus Coagulans Cabicy-Cassie Spores Medium composition storage duration
(month) Spore Population (cfu / ml) B. coagulans KACC 10117 B. coagulans KBC-KC1 Nutrition badge 0 6.8 × 10 ⁴ 7.9 × 10 ⁴ One 4.1 × 10 ³ 4.9 × 10 ³ 3 8.5 × 10 ² 1.1 x 10 ³ 6 3.1 × 10 ² 9.6 × 10 ² Nutritional Medium + Jujube Hot Water Extract 10% 0 4.7 × 10 ⁴ 8.6 × 10 ⁶ One 2.1 x 10 ³ 5.4 × 10 ⁶ 3 9.8 × 10 ² 3.0 × 10 ⁶ 6 3.4 x 10 ² 9.6 × 10 ⁵ Nutritional Medium + Jujube Ethanol Extract 10% 0 3.9 × 10 ⁴ 8.4 × 10 ⁶ One 1.0 x 10 ³ 3.1 × 10 ⁶ 3 8.8 × 10 ² 1.2 × 10 ⁶ 6 5.1 x 10 ² 7.5 × 10 ⁵

< Manufacturing example  1> Preparation of feed additive

The inventors of the invention Bacillus Coagulance Cabisi-Cassie 1 ( Bacillus coagulans KBC-KC1) strain, the culture medium of the strain or spores formed from the strain as an active ingredient was prepared a feed additive with the following composition.

Bacillus coagulase Cabisi-Cassie 1 strain, culture of the strain or spores formed from the strain: 0.1 to 10% by weight, prepared hot water extract or ethanol jujube extract: 0.1 to 10% by weight, lipolysis Lipase: 0.001 to 0.01% by weight, tricalcium phosphate: 1 to 20% by weight, vitamin E: 0.01 to 0.1% by weight, enzyme powder: 1 to 10% by weight, lactic acid bacteria: 0.1 to 10% Parts by weight, glucose: 20 to 90% by weight was added to the mixer to mix to prepare a feed additive.

< Manufacturing example  2> Preparation of Lactic Acid Bacteria Formulations

The inventors aseptically inoculated Bacillus coagulans KBC-KC1 strain into MRS medium containing 1, 5 and 10% by weight of hot water extract or ethanol extract, followed by 37 to 24 Incubated for hours. Thereafter, 700% by weight of the excipient (zeolite or cereals and by-products thereof) of the lactic acid bacteria culture medium was added to the mixer, and the lactic acid bacteria culture medium was sprayed while stirring at 30 RPM to prepare a lactic acid bacteria culture medium adsorbate. In addition, 1200% by weight of rice hull powder of the lactic acid bacteria culture medium adsorbate was added to the mixer to mix and solid culture for 5 days at a rate of 35 RPM to prepare a lactic acid bacteria formulation.

Bacillus coagulase Cabici-Cassie 1 strain of the present invention forms spores having excellent heat resistance and storage stability when the jujube extract is included in the culture medium, a novel livestock feed probiotic, feed additives, pharmaceutical composition for enhancing livestock immunity and lactic acid bacteria It can be usefully used for the development of the formulation.

Agricultural Genetic Resources Information Center KACC91677P 20110921

Attach an electronic file to a sequence list

Claims (8)

1) Bacillus coagulans KBC-KC1 strain 1 to 10 weight relative to the total weight of the medium, characterized in that having the 16S rRNA sequence deposited as Accession No. KACC91677P and described in SEQ ID NO: 1 Culturing in a medium containing jujube extract added in%; And
2) probiotic from Bacillus coagulans KBC-KC1 strain comprising the step of obtaining the strain, the culture of the strain or the spores formed from the strain cultured in step 1) How to make.
delete The method of claim 1, wherein the culturing of step 1) is a probiotic production method, characterized in that the culture for 3 to 7 days at 20 to 50 ℃.
Feed additive containing the probiotics composition produced by claim 1 as an active ingredient.
A lactic acid bacterium preparation containing the probiotics composition produced by claim 1 as an active ingredient.
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