KR20170108214A - Bacillus subtilis hd 9098, probiotics composition including the same and method of manufacturing thereof - Google Patents

Abstract

The present invention relates to a feed additive having improved immunity enhancing effect of spent red ginseng by using a Bacillus subtilis strain, and more specifically, to a method of preparing a liquid probiotic composition comprising: a fermented culture fluid preparation step of fermenting at room temperature a culture fluid of Bacillus subtilis HD 9098 (Deposit No. KCTC18456P) containing spent red ginseng as a fermentation source; a separated solution preparation step of centrifuging the fermented culture fluid to remove solids therefrom; and a liquid probiotic agent preparation step of adding one or more types of organic acids and gum-based compounds to the separated solution to thereby form a liquid preparation. Accordingly, the present invention may provide a method of preparing a probiotic composition and a liquid probiotic composition prepared by the method, in which viable cell count, crude saponin content, ginsenocide content, conversion ratio of spores to bacterial cells, antibacterial property, and antioxidant function are improved; and when fed to livestock, the effects in terms of excellent intestinal regulation, immunity enhancement, improvement of feed efficiency, and improvement of livestock house environment can be achieved; and storage stability is improved, and production cost can be lowered due to being suitable for mass-production. Additionally, a liquid probiotic agent of the present invention ferments spent red ginseng, which is a by-product of red ginseng, with Bacillus subtilis HD 9098, thereby further enhancing ginsenoside concentration. Such a liquid probiotic agent may be beneficially used in a feed additive having immunity regulatory effect and veterinary medications.

Description

TECHNICAL FIELD The present invention relates to a Bacillus subtilis HD 9098 strain having excellent red ginseng rooting efficacy, a prophylactic composition for immunity enhancement containing the same, and a method for producing the same.

The present invention relates to a Bacillus subtilis HD 9098 strain having excellent red ginseng rooting efficacy, a prophylactic composition for immunity enhancement containing the same, and a preparation method thereof.

Since 2005, the Ministry of Food, Agriculture, Forestry and Fisheries has promoted the policy of reducing antibiotics for the production of compound feeds. As a result, the use of antibiotics in the formulated diets was totally prohibited from July 2011 due to the amendment of notification related to the Feed Control Act, due to the scope and criteria of hazardous feed (Ministry of Agriculture, Forestry and Fisheries Notification No. 2010-142, 2010.12.27).

In order to cope with such a policy, a method of feeding feedstuffs that can replace antibiotics to feedstuffs has been used, but these feedstocks are highly dependent on imports and have a disadvantage in that they are not easy to generate profits due to their high cost. These economic difficulties have led to the deterioration of the management balance of livestock farmers.

In addition, stench in the housing during massive breeding of livestock and densely packed breeding is another factor that increases the difficulty of livestock farming management. The cause of stench in house is mainly harmful gas and odor causing substance generated by microorganisms in manure.

Examples of stench-inducing substances in houses include ammonia, trimethylamine, hydrogen sulfide and the like. These odor-causing substances pollute the soil and the atmosphere and cause physical and mental damages to the people living in the surrounding areas, thus causing complaints about the operation of the housing. The Ministry of Environment is continuously strengthening standards for livestock manure and odor emission from livestock farmers through the Anti-Odor Control Law and the Recommendation for Specified Criteria for Livestock Restricted Areas.

In recent years, due to the changes in regulations on the two-axis industry, the antibiotics for livestock have been replaced by feed additives such as environmentally friendly raw materials to reduce immunity to bacteria and realize eco- There is an increasing demand for the development of technologies that can meet demand.

As an alternative to such a market demand, there is known a technique of feeding red ginseng roots mixed with livestock compound feeds with low industrial utilization. However, in the conventional red ginseng roots mixed feed, since the active ingredient is already separated by hot water and ethanol extraction and then dried, the absorption efficiency of the useful ingredient is very low when it is fed to the livestock, There is a dot.

Another conventional technique is known in which red ginseng roots are fermented with microorganisms and used as feed ingredients. However, this method is also insufficient in the effectiveness of the useful ingredient when fed to livestock, and conventional microorganisms are insufficient in the number of bacteria due to inhibition of cultivation by the available nitrogen-free, crude fiber and crude protein of red ginseng roots, A medium containing specific components must be separately added, and there is a need to additionally perform a process of artificially liberating saponin.

One object of the present invention is to provide a method for the production of Bacillus subtilis HD 9098 (hereinafter referred to as " Bacillus subtilis HD 9098 "), which has probiotic activity, pathogenic microbial antimicrobial activity and red ginseng pulping efficacy and has an effect of increasing crude saponin content and ginsenoside liberation rate bacillus subtilis HD 9098, KCTC18456P).

Another object of the present invention is to provide a method for producing an extract of the present invention which is excellent in viable cell count, crude saponin content, ginsenoside content, bacterial spore conversion rate, antimicrobial property, antioxidant property and antifouling property, , A feedstock efficiency improvement and a housing environment improvement effect, and is excellent in storage stability, and a method for producing the liquid probiotics composition which is suitable for mass production and has an effect of lowering the manufacturing cost.

One embodiment of the present invention relates to a method for producing Bacillus subtilis HD 9098 (hereinafter, referred to as " Bacillus subtilis HD 9098 "), which has probiotic activity, pathogenic microbial antibacterial activity and red ginseng pulping efficacy and has an effect of increasing crude saponin content and ginsenoside liberation rate bacillus subtilis HD 9098, KCTC 18456P).

Another embodiment of the present invention is a method for producing a fermentation broth comprising fermenting a red ginseng broth by using a culture solution of Bacillus subtilis HD 9098 (bacillus subtilis HD 9098, KCTC 18456P); A separation liquid preparation step of removing the solid content from the fermentation culture liquid; And a liquid prophylactic agent preparation step in which at least one of organic acid and gum compounds is added to the separated liquid to form a liquid; And a method for producing a liquid probiotics.

In the fermentation broth preparation step, Bacillus subtilis HD 9098 (Bacillus subtilis HD 9098, KCTC 18456P) was inoculated on a medium containing 0.01% to 50% by weight of red ginseng on the basis of a 5 L to 5000 L fermenter, , And subjecting the culture broth to a liquid fermentation at a temperature of 15 ° C to 45 ° C for 6 to 120 hours by adjusting the aeration amount.

The step of producing the liquid probiotics is performed by adding 0.01 to 50.0% by weight of organic acid, 0.001 to 10.0% by weight of gum compounds and 0.01 to 80% by weight of distilled water to 10.0 to 99.90% Lt; / RTI >

The organic acid includes at least one of citric acid, acetic acid and the like, and the gum compounds include at least one of xanthan gum, guar gum and gellan gum. can do.

Another embodiment of the present invention relates to a liquid prophylactic composition prepared by the above-described method for producing a liquid probiotic composition.

The liquid prophylactic composition may be selected from the group consisting of Bacillus subtilis HD 9098, KCTC 18456P strain, cellulase, amylase, lipase, protease, pedctinase, crude saponin , Ginsenoside Rb1, ginsenoside Rg1, and ginsenoside Rg3.

The liquid probiotic composition may have a viable cell viability of 90% or more and a crude saponin survival rate of 95% or more after an accelerated condition test in which the liquid probiotic composition is allowed to stand for 4 weeks at a temperature of 40 ± 1 ° C and a relative humidity of 75 ± 5%.

Bacillus subtilis HD 9098 (bacillus subtilis HD 9098), which has probiotic activity, pathogenic microbial antimicrobial activity and red ginseng rooting efficacy, and has the effect of increasing crude saponin content and ginsenoside liberation rate during fermentation of red ginseng roots , KCTC18456P), and by including the culture medium of the strain, the number of viable cells, crude saponin content, ginsenoside content, bacterial spore conversion rate, antimicrobial property, antioxidant and antifouling property are improved, the precipitate production rate is extremely low, To provide a liquid type probiotic composition capable of realizing excellent suits, immunity enhancement, feed efficiency improvement and housing environment improvement effect, improving storage stability, being suitable for mass production and lowering the manufacturing cost, and a method for producing the same have.

Fig. 1 shows growth curves and spore conversion rates of strains prepared in Preparation Example 2 of the present invention.
2 shows the result of measurement of the enzyme fraction ratio of the strain prepared in Preparation Example 2 of the present invention.
Fig. 3 shows the results of evaluation of the E. coli ATCC 10536 inhibitory ability of the strain prepared in Preparation Example 2 of the present invention.
Fig. 4 shows the results of evaluation of the inhibitory ability of Salmonella choleraesuis ATCC 10708 of the strain prepared in Preparation Example 2 of the present invention.
FIG. 5 shows the results of evaluation of the inhibitory ability of Salmonella enteritidis ATCC 13076 of the strain prepared in Preparation Example 2 of the present invention.

One embodiment of the present invention relates to a method for producing Bacillus subtilis HD 9098 (hereinafter, referred to as " Bacillus subtilis HD 9098 "), which has probiotic activity, pathogenic microbial antibacterial activity and red ginseng pulping efficacy and has an effect of increasing crude saponin content and ginsenoside liberation rate bacillus subtilis HD 9098, KCTC 18456P).

In addition, embodiments of the present invention can use the above-described Bacillus subtilis HD 9098 (KCTC18456P) strain or a culture solution of the strain as a liquid probiotic. Accordingly, the present invention provides a method for producing a microorganism which is excellent in viable cell count, crude saponin content, ginsenoside content, bacterial spore conversion rate, antimicrobial activity, antioxidant and antifouling property, very low sediment production rate, It is possible to provide a liquid prophylactic agent excellent in storage stability and a method of producing the liquid prophylactic composition capable of lowering the manufacturing cost by being suitable for mass production, which is industrially useful.

The bacillus subtilis HD 9098 (HD 9098) strain of the present invention has an efficacy against the red ginseng root, and even when the red ginseng root is used as the fermentation source, the strain is excellent in the strain. The decomposing enzyme produced from the strain has an excellent effect of decomposing the fiber of the red ginseng roots and including the ginsenoside-containing immunostimulating substance in the fermentation broth.

Specifically, when the Bacillus subtilis HD 9098 (bacillus subtilis HD 9098, KCTC 18456P) strain was applied to a liquid type probiotic composition using the red ginseng roots as a fermentation source, the cultivation of the strain and the fermentation of red ginseng roots proceed simultaneously, The present invention realizes an effect of increasing the content of useful components such as crude saponin, ginsenoside Rb1, Rg1, and Rg3 by implementing excellent fermentation while omitting the hassle of adding a specific ingredient to the medium to proceed with the fermentation .

Thus, the Bacillus subtilis HD 9098 (KCTC18456P) strain of Bacillus subtilis HD 9098 can ferment a red ginseng product, which is a by-product of red ginseng, with excellent efficiency, thereby providing a probiotic agent that realizes excellent immunity enhancement with low production cost have. Such a liquid prophylactic agent can be particularly usefully applied to feed additives and animal drugs having an immunomodulating effect.

In one embodiment, the liquid probiotics to which the Bacillus subtilis HD 9098 strain is applied may be excellent in the immune enhancement effect on industrial animals, particularly piglets.

The Bacillus subtilis HD 9098 strain of the present invention has an excellent probiotic activity such as suitability and polymer degradability, thus realizing the effect of improving the dressing, intestinal microflora, immunity, and feed efficiency as a probiotic agent.

The Bacillus subtilis HD 9098 strain has excellent secretion ability of cellulase, amylase, lipase, protease and pectinase. As a result, the culture of Bacillus subtilis HD 9098 strain was inhibited by soluble nitrogen-free water, crude fiber and crude protein contained in more than 90% of red ginseng roots, and thus the effect of increasing viable cell count can be excellent.

In one embodiment, the Bacillus subtilis HD 9098 strain may be better able to secrete alpha -amylase and protease. In this case, the foot efficiency for red ginseng roots and the dressing effect for economic animals can be more excellent.

The Bacillus subtilis HD 9098 strain is excellent in pathogenic microbial antimicrobial activity, and can control the microbial total bacterial count in the feces of the livestock and inhibit the growth rate of E. coli and intestinal bacteria. In addition, the Bacillus subtilis HD 9098 strain can improve the housing environment when the probiotic agent is sprayed into the stomach during the feeding process through the pathogenic microbial antibacterial activity.

In one embodiment, the Bacillus subtilis HD 9098 strain has an inhibitory potency against E. coli ATCC 10536, Salmonella choleraesuis ATCC 10708 and Salmonella enteritidis ATCC 13076 Particularly excellent. In such a case, the effect of suiting the economic animal and the effect of enhancing the immunity can be more excellent.

The excellent intestinal flora control effect, enzyme activity and harmful bacteria inhibitory effect of the Bacillus subtilis HD 9098 strain due to the probiotic activity and the pathogenic microbial antimicrobial activity of the Bacillus subtilis HD 9098 strain were evaluated as a probiotic agent, The effect of improving the housing environment can be realized.

The Bacillus subtilis HD 9098 (bacillus subtilis HD 9098, KCTC 18456P) strain of the present invention has an excellent spore conversion rate as compared to other Bacillus subtilis strains. Thus, the Bacillus subtilis HD 9098 strain (bacillus subtilis HD 9098, KCTC18456P) has an effect of further enhancing the spore content and storage stability of the liquid probiotic.

In the present invention, in order to identify the microorganisms which can be used for increasing the crude saponin content and the ginsenoside liberation ratio during fermentation of red ginseng roots, the chungkukjang was suspended in sterilized saline, diluted, laid on NA agar medium, The colony formed after the time culture was subcultured to isolate the strains having excellent probiotic activity, pathogenic microorganism antibacterial activity and red ginseng thinning efficacy. The strain thus isolated has been identified as a new strain belonging to Bacillus subtilis. The present inventors named the strain thus selected as Bacillus subtilis HD 9098, and the microorganism resource center of Korea Biotechnology Research Institute 8, and was granted accession number KCTC18456P.

The Bacillus subtilis HD 9098 strain is a strain isolated from Chungkukjang. It is a non-pathogenic search experiment using blood agar and a livestock activity search experiment for amylase, protease, cellulase, lipase, protease and pectinase Active).

The Bacillus subtilis HD 9098 strain was confirmed to be a Pseudomonas strain having 98.6% or more homology with Bacillus subtilis through phylogenetic analysis, morphological analysis and physiological characteristics analysis.

Phylogenetic characterization was performed by 16S rDNA sequence analysis and PCR. This 16S rDNA sequence analysis was performed under the same conditions as described in Production Example 1 described later.

Another embodiment of the present invention is a method for producing a fermentation broth comprising fermenting a red ginseng broth by using a culture solution of Bacillus subtilis HD 9098 (bacillus subtilis HD 9098, KCTC 18456P); A separation liquid preparation step of removing the solid content from the fermentation culture liquid; And a liquid prophylactic agent preparation step in which at least one of organic acid and gum compounds is added to the separated liquid to form a liquid; And a method for producing a liquid probiotics.

The method of producing the liquid probiotics according to the present invention uses red ginseng roots as the fermentation source of Bacillus subtilis HD 9098 (HD 9098), which has excellent suitability and polymer decomposition activity, The present invention provides an effect of decomposing the fiber of the red ginseng roots and including a high content of an immunostimulating substance including ginsenoside contained in the red ginseng roots in the fermentation broth.

Specifically, the method for producing a liquid probiotics according to the present invention comprises culturing the strain of Bacillus subtilis HD 9098 (bacillus subtilis HD 9098, KCTC 18456P) in a medium containing red ginseng roots and simultaneously culturing the strain and fermenting red ginseng roots, It is possible to omit the hassle of adding a specific ingredient to the medium for proceeding with the fermentation of red ginseng roots and to achieve an excellent fermentation degree.

In the fermentation broth preparation step, Bacillus subtilis HD 9098 (Bacillus subtilis HD 9098, KCTC 18456P) was inoculated on a medium containing 0.01% to 50% by weight of red ginseng on the basis of a 5 L to 5000 L fermenter, , And subjecting the culture broth to a liquid fermentation at a temperature of 15 ° C to 45 ° C for 6 to 120 hours by adjusting the aeration amount.

In one embodiment, the step of preparing the fermentation broth comprises inoculating Bacillus subtilis HD 9098 (Bacillus subtilis HD 9098, KCTC 18456P) into a medium containing red ginseng root on the basis of a Jar fermenter of 5 L to 500 L, After the culture medium is prepared, the above culture broth is subjected to liquid fermentation and culture at pH 5.6 to pH 7.5, DO 1 ppm to 20 ppm, 35 ° C to 37 ° C and aeration amount of 1 NL / min to 4 NL / min for 6 hours to 54 hours ≪ / RTI > The liquid probiotics prepared in the liquid fermentation and culturing conditions showed that the viable cell count, spore conversion, crude saponin content, ginsenoside Rb1 increase rate by fermentation, increase rate of ginsenoside Rg1 by fermentation, and increase rate of ginsenoside Rg3 by fermentation Can be improved.

In the preparation step of the fermentation broth, the content of the red ginseng root in the medium may be 0.01 wt% to 50.0 wt% of the red ginseng.

In one embodiment, the step of preparing the fermentation broth may comprise 5% to 12.5% by weight of red ginseng. Within the above range, the efficiency in which the amount of the red ginseng powder added to the input amount of the red ginseng powder is increased can be increased. Such an effect is more advantageous in increasing profit and loss when the method of producing liquid probiotics of the present invention is applied to mass production.

The size of the ginseng roots can be controlled by a pretreatment process before being added to the medium. For example, the red ginseng roots can be put into the medium after controlling the particle size to 1,000 μm or less, 850 μm or less, 500 μm or less, 300 μm or less or 100 μm or less. In this case, it is possible to increase the pumping efficiency with respect to the amount of red ginseng powder, and further improve the spore conversion rate and crude saponin content.

The method for producing a liquid probiotics according to the present invention carries out the step of preparing a separated liquid for removing solid content from the fermentation broth prepared as described above. The method for removing the solid content may include, for example, centrifugation, filtration, compression, and the like.

For example, the separation liquid preparation step may include centrifuging the fermentation broth at 3000 rpm to 5000 rpm, separation rate of 150 mL / min to 250 mL / min, and removing the precipitate. The liquid probiotics prepared under the conditions for preparing the separated liquid can increase the survival rate of live cells and the recovery rate of crude saponin, and at the same time, the storage stability can be further improved because no precipitates are formed even during long-term storage.

In the method for producing a liquid probiotics according to the present invention, the step of preparing a liquid probiotics is carried out by adding at least one of organic acid and gum compounds to the separated liquid prepared as described above and liquefying it. In addition, the separated solution may be diluted to a desired concentration through distilled water or the like depending on the case, and may further include a suitable additive for a probiotic agent to improve the desired efficacy.

The step of producing the liquid probiotics is performed by adding 0.01 to 50.0% by weight of organic acid, 0.001 to 10.0% by weight of gum compounds and 0.01 to 80% by weight of distilled water to 10.0 to 99.90% Lt; / RTI >

The organic acid may include, for example, at least one of citric acid, acetic acid and the like, and the gum-type compound may be, for example, xanthan gum, guar gum, gellan gum), and the like. In such a case, it is possible to provide a probiotic agent with excellent stability even when the viable cell water survival rate and crude saponin residual rate are high, and the contamination does not occur and storage is carried out for a long period in a liquid phase.

In one embodiment, the step of producing the liquid probiotics comprises adding from 0.05% to 0.5% by weight of organic acid and from 0.05% to 1.00% by weight of gum compounds to 98.5% to 99.90% , The organic acid includes at least one of citric acid and acetic acid and the gum compound includes at least one of xanthan gum, guar gum and gellan gum . In this case, even after the accelerated condition test in which the liquid probiotic composition is allowed to stand at 40 ± 1 ° C and 75 ± 5% for 4 weeks, the storage stability of the probiotics is improved even when the liquid probiotic composition is stored for a long period of time .

As described above, the method of the present invention for producing a liquid probiotics according to the present invention can be applied not only to liquid fermentation but also to liquify (formulate into a liquid form) And the body absorption rate can be further increased. In this case, it can be used as a feed additive because it can exert a sufficient effect even with a small amount of feed compared to conventional red ginseng roots mixed feed, and it is possible to further increase productivity and profitability of the two axes.

Another embodiment of the present invention relates to a liquid prophylactic agent prepared according to the aforementioned method for producing a liquid probiotics.

The liquid probiotic composition was prepared from a culture solution of Bacillus subtilis HD 9098 (KCTC18456P) strain and red ginseng roots as a fermentation source, and the metabolites produced by the strain and useful components liberated by the strain from red ginseng roots . ≪ / RTI >

Specifically, the liquid probiotic composition may be selected from the group consisting of strains of Bacillus subtilis HD 9098, KCTC 18456P, cellulase, amylase, lipase, protease, pedctinase, , Crude saponin, ginsenoside Rb1, ginsenoside Rg1, and ginsenoside Rg3. Thus, the liquid probiotics composition can be fed to the livestock to achieve excellent suitability, immunity enhancement, feed efficiency improvement and housing environment improvement effect, and the storage stability can be improved.

In one embodiment, the liquid probiotic composition may comprise a strain of Bacillus subtilis HD 9098 and secreted alpha-amylase and protease thereby.

In addition, the liquid probiotic composition has antimicrobial properties that suppress the total microbial count of microorganisms in the feces of economic animals and the rate of growth of coliform bacteria and intestinal bacteria. The intestinal flora control effect and the excellent enzyme activity power can reduce the odor-inducing substances and harmful substances in the livestock feces when fed to the animals, thereby improving the housing environment.

In one embodiment, the liquid probiotic composition can be particularly effective in inhibiting E. coli ATCC 10536, Salmonella choleraesuis ATCC 10708 and Salmonella enteritidis ATCC 13076 .

In addition, the liquid probiotic composition has an excellent spore conversion rate of Bacillus subtilis HD 9098 (KCTC 18456P). Thus, the liquid probiotics can be more excellent in storage stability.

Specifically, the liquid type prophylactic composition does not cause contamination even after the accelerated condition test in which the temperature is 40 ± 1 ° C. and the relative humidity is 75 ± 5% for 4 weeks, thereby improving the storage stability of the probiotics .

In one embodiment, the liquid probiotic composition has a survival rate of 90% or more and a survival rate of crude saponin of 95% or more after the accelerated condition test in which the liquid probiotic composition is allowed to stand at 40 ± 1 ° C and 75 ± 5% Or more.

Accordingly, the present invention provides a method for producing a microorganism which is excellent in viable cell count, crude saponin content, ginsenoside content, bacterial spore conversion rate, antimicrobial activity, antioxidant property and antifouling property, , A feed efficiency improvement and a housing environment improvement effect can be realized, and a liquid probiotic composition excellent in storage stability can be provided.

Example

Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to preferred embodiments of the present invention. It should be understood, however, that this is provided as illustrative of the present invention and is not to be construed as limiting the invention in any way.

The contents not described here are sufficiently technically inferior to those skilled in the art, and a description thereof will be omitted.

Manufacturing example  1. Selection and Identification of Strain

Five kinds of chungkukjang which were collected in the traditional market were suspended in sterilized saline solution of 5 g each, and then they were spread on NA agar medium and cultured at 36 ± 1 ° C for 24 hours. The colonies formed after culturing were separated and cultured for 3 generations to isolate Bacillus subtilis strains pure. The pure strains were plated on agar plates supplemented with Soluble Starch (3%) or carboxymethyl cellulose (3%) and cultured at 36 ± 1 ° C for 24 hours to select the strain Bacillus subtilis HD 9098 Respectively.

<Identification of Strain>

1) Analysis of systematic characteristics

DNA for 16S rDNA sequence analysis was extracted from the above-selected strain (Bacillus subtilis HD 9098), and PCR was performed using 27F and 1492R primers. The PCR was electrophoresed to confirm that a PCR product was obtained. The PCR product identified above was purified and analyzed by DNA analysis agency (Solgent). As a result, it was confirmed that it had 99% homology with Bacillus subtilis.

Based on these results, we have analyzed the ndetic tree based on the 16S rDNA sequence. As a result, the previously selected strains (Bacillus subtilis HD 9098, KCTC18456P) belong to B. subtilis (subsp. Subtilis) Respectively.

2) Morphological characterization

Gram staining was performed on the strain selected above. As a result, the strain (Bacillus subtilis HD 9098) was identified as a gram positive strain.

In addition, the strain selected above was observed by scanning electron microscope (SEM) and its morphology was observed. As a result, it was confirmed that the strain was a typical bacillus strain shape in the form of a bacterium of about 3 to 4 쨉 m.

3) Analysis of physiological characteristics

The strain selected above was cultured to obtain a single colony. The strain was suspended in API 50 CHB medium and adjusted to a suspension of 2 McFarland to prepare a strain suspension. The strain suspension was added to an API 50 CH strip kit and after 24 hours, the activity was confirmed. The confirmation of the active state was observed through the color change of the phenol red indicator, and it was confirmed that it turned yellow. The results of the changes of the kit were recorded on a recording sheet and entered into the web site (http://apiweb.biomerieux.com) to analyze the physiological characteristics and identify the strains of the selected strain (Bacillus subtilis HD 9098). As a result of this identification, it was confirmed that the homology with Bacillus subtilis was 98.6%, and the results are shown in Table 1 (+: positive, -: negative).

number Carbohydrates Glucose utilization number Carbohydrates Glucose utilization 0 Control - 25 Esculine + One Glycerol + 26 Salicine + 2 Erythritol - 27 Cellobiose + 3 D-Arabinose - 28 Maltose + 4 L-Arabinose + 29 Lactose + 5 Ribose + 30 Melibiose - 6 D-Xylose + 31 Saccharose + 7 L-Xylose - 32 Trehalose + 8 Adonitol - 33 Inuline - 9 β-Methyl-xyloside - 34 Melezitose - 10 Galactose - 35 D-Raffinose - 11 D-Glucose + 36 Amidon - 12 D-Fructose + 37 Glycogene - 13 D-Mannose + 38 Xylitol - 14 L-Sorbose - 39 β-Gentiobiose + 15 Rhamnose - 40 D-Turanose - 16 Dulcitol - 41 D-Lyxose - 17 Inositol + 42 D-Tagatose - 18 Mannitol + 43 D-Fucose - 19 Sorbitol + 44 L-Fucose - 20 alpha -Methyl-D-mannoside - 45 D-arabitol - 21 alpha -Methyl-D-glucoside + 46 L-Arabitol - 22 N-Acetyl glucosamine - 47 Gluconate - 23 Amygdaline + 48 2-Ceto-gluconate - 24 Arbutine + 49 5-Ceto-gluconate -

Manufacturing example  2. Preparation of sample for evaluation of efficacy of strain

In order to examine the optimal growth conditions of B. subtilis HD 9098 strain selected in Preparation Example 1, the culture conditions under the physicochemical conditions such as incubation time, culture temperature, initial pH, carbon source, nitrogen source and inorganic salts were examined.

TSB medium (Tryptic soy broth) Bacillus subtilis HD 9098 strain, which had been sliced into a 100 ml Erlenmeyer flask containing 20 ml of the culture medium, was inoculated once with platinum at a temperature of 35 ± 1 ° C for 24 hours, Was used as a sample for evaluating strain efficacy. The degree of growth of each sample was diluted by decidual dilution method, and then plated on a Tryptic soy agar medium and cultured at 37 ° C for 24 hours. The number of emerging colonies was counted to determine the growth rate (CFU / ml) of each strain.

The optimum conditions for the growth of the strain were determined as the concentration (%, W / V), and the optimum conditions for the growth of the strain were as follows: the incubation time, the incubation temperature, the initial pH, the carbon source effect, as it comprised a Glucose 0.5%, Corn Flour 0.5% , 0.5% Soybean Flour, NaCl 0.1%, K 2 HPO 4 0.1%, MgSO 4.7 H 2 O 0.05% and MnSO _4.7 H ₂ O 0.005%.

As a result of culturing Bacillus subtilis HD 9098 under the above conditions for 48 hours, the number of viable cells per 1 ml of culture was 1 to 7E + 09 cfu.

&Lt; Evaluation of efficacy of strain &

1) Evaluation of spore conversion power

FIG. 1 shows the result of measuring the growth curve of the strain and the spore transformation ability in the culture medium of Preparation Example 2 prepared as described above.

2) Determination of starch hydrolysis power (α-Amylase) and protein digestive capacity (protease) activity

In the culture medium of Preparation Example 2 prepared above, crude enzyme solution was collected from each culture time to analyze enzyme secretion potency (starch glycation potency and protein digestibility). The starch sugar strength and protein digestibility were measured according to the Digestibility Test in the General Test Methods for Veterinary Medicines, Article 5.

The results showed that starch hydrolysis power and protein digestibility of Bacillus subtilis HD 9098 strain increased during the growing stage when the growth of the strain became vigorous and the increase was small when the growth of the strain was stagnated. The result of this enzyme secretion evaluation is shown in Fig.

3) Evaluation of pathogenic microorganism (harmful bacteria) antimicrobial activity of the strain

The inhibitory potency of pathogenic microorganisms (noxious bacteria) (E. coli ATCC 10536, Salmonella choleraesuis ATCC 10708, Salmonella enteritidis ATCC 13076) in the culture medium of Preparation Example 2 was measured. Tryptic soy broth was inoculated at a concentration of 10,000 cfu / ml for each of the harmful bacteria, and 500 and 1000-fold dilutions of the previously prepared culture were inoculated and cultured at 36 ± 1 ° C for 72 hours. After 24, 48 and 72 hours incubation, E. coli was cultivated in Chromocult Agar (Merck) medium and Salmonella spp. In Salmonella Shigella Agar (Difco) medium after decidual dilution and cultured at 36 ± 1 ℃ for 75 hours. As a result, it was shown that 99.5% of all three harmful bacteria were inhibited at all concentrations. The results of the analysis are shown in FIGS.

FIG. 3 shows the result of evaluation of the inhibitory ability of E. coli ATCC 10536 of the strain of Preparation Example 1, FIG. 4 shows the results of evaluation of the inhibitory effect of Salmonella choleraesuis ATCC 10708 of the strain of Preparation Example 1, FIG. 5 shows the results of evaluation of the inhibitory ability of Salmonella enteritidis ATCC 13076 of strain 1 of the present invention.

4) Measuring crude saponin conversion ability of selected strain

The strains selected in Preparation Example 1 were cultured for 24, 48, and 72 hours in a culture medium containing 5% red ginseng in an incubator (35 rpm, 1 rpm, 100 rpm) for 24, 48 and 72 hours, And spore conversion were evaluated. Table 2 shows the results of evaluating the efficacy of the strain on viable cell count, spore conversion rate and crude saponin content.

Preparation Example 1
Culture progress 0 hours 24 hours 48 hours 72 hours Number of viable cells (CFU / ml) 8.90E + 06 8.70E + 08 9.90E + 08 1.12E + 09 Spore Conversion Rate (%) - 8.7 67.5 80.2 Crude saponin content (%) 0.18 0.24 0.41 0.51

Example  1 to 5

Red ginseng roots were pulverized and sorted by particle size using a laboratory mesh to prepare a medium containing 10% red ginseng. Fermentation was carried out on the medium using Bacillus subtilis HD 9098 strain isolated in Preparation Example 1.

The cells were cultured for 48 hours at an incubation temperature of 35 짹 1 째 C and at an agitation (100 rpm) incubator, and the viable cell count and crude saponin content were measured and shown in Table 3 below.

Example 1 Example 2 Example 3 Example 4 Example 5 Red ginseng
Content (% by weight) 10 10 10 10 10 Minimum particle size (탆) Exceeding 1000 Less than 850 Less than 500 Less than 300 - Maximum particle size (탆) - 500 or more 300 or more 100 or more Less than 100 0 hour culture

Viable cell count
(cfu / mL) 8.90E + 06 8.80E + 06 1.01E + 09 9.10E + 08 9.20E + 08 Spore Conversion Rate (%) - - - - - Crude saponin content (%) 0.17 0.17 0.17 0.17 0.17 Culture for 48 hours

Viable cell count
(cfu / mL) 8.30E + 08 8.70E + 08 9.10E + 08 1.10E + 09 1.04E + 09 Spore Conversion Rate (%) 35.4 45.8 49.5 61.2 70.2 Crude saponin content (%) 0.27 0.28 0.32 0.35 0.45

Example  6 to 9

Red ginseng roots were pulverized and sorted by particle sizes selected using a laboratory mesh, and then a medium containing 10% red ginseng was prepared in a 5 L class Jar fermenter. Fermentation was carried out on the medium using Bacillus subtilis HD 9098 strain isolated in Preparation Example 1. The culture conditions were changed as shown in Table 4 below.

The cells were cultured for 48 hours at a culture temperature of 36 占 폚 and a stirring (100 rpm) incubator, and the viable cell count and crude saponin content were measured and shown in Table 4 below.

Example 6 Example 7 Example 8 Example 9 Red ginseng Content (% by weight) 10 10 10 10
Culture conditions

pH 5.6 to 7.5 5.6 to 7.5 5.6 to 7.5 5.6 to 7.5 DO (ppm) 1 to 20 1 to 20 1 to 20 1 to 20 RPM 100 200 250 200 Temperature (℃) 36 ± 1 36 ± 1 36 ± 1 36 ± 1 Air flow rate (NL / min) One 2 4 2
result
Viable cell count 1.10E + 09 1.56E + 09 2.20E + 09 1.35E + 09 Spore Conversion Rate (%) 75.8 78.6 86.0 75.6 Crude saponin content (%) 0.41 0.46 0.49 0.44

Example  10 to 12

Red ginseng roots were pulverized, and selected by particle size using a laboratory mesh. Then, in a 50 L Jar fermenter, a medium containing 12.5% red ginseng was prepared. Fermentation was carried out on the medium using Bacillus subtilis HD 9098 strain isolated in Preparation Example 1. The culture conditions were changed as shown in Table 5 below.

The cells were cultured for 48 hours at a culture temperature of 36 占 폚 and a stirring (100 rpm) incubator, and the viable cell count and crude saponin content were measured and shown in Table 4 below.

Example 10 Example 11 Example 12 Red ginseng Content (% by weight) 12.5 10 10
Culture conditions

pH 5.6 to 7.5 5.6 to 7.5 5.6 to 7.5 DO (ppm) 1 to 200 1 to 200 1 to 200 RPM 100 200 250 Temperature (℃) 36 ± 1 36 ± 1 36 ± 1 Air flow rate (NL / min) One 2 4
result
Viable cell count 1.10E + 09 2.67E + 09 2.61E + 09 Spore Conversion Rate (%) 81.2 81.2 80.9 Crude saponin content (%) 0.48 0.58 0.62

Example  13 to 15

Red ginseng roots were pulverized and sorted by particle sizes selected using a laboratory mesh, and a medium containing 12.5% red ginseng was prepared in a 500 L Jar fermenter. Fermentation was carried out on the medium using Bacillus subtilis HD 9098 strain isolated in Preparation Example 1. The culture conditions were changed as shown in Table 5 below.

The cells were cultured at a culture temperature of 36 ± 1 ° C for 48 hours in an agitation (100 rpm) incubator, and the viable cell count and crude saponin content were measured and shown in Table 6 below.

Example 13 Example 14 Example 15 Red ginseng Content (% by weight) 12.5 10 10
Culture conditions

pH 6.5 to 7.5 6.5 to 7.5 6.5 to 7.5 DO (ppm) 1 to 200 1 to 200 1 to 200 RPM Variance Variance Variance Temperature (℃) 36 ± 1 36 ± 1 36 ± 1 Air flow rate (NL / min) Variance Variance Variance
result
Viable cell count 3.20E + 09 1.98E + 09 2.41E + 09 Spore Conversion Rate (%) 81.2 86.7 82.4 Crude saponin content (%) 0.72 0.65 0.66

Example  16 to 19

The centrifugation conditions of the culture solution prepared in Example 12 were changed to remove the precipitate and prepare a separation liquid. The centrifugation conditions were changed as shown in Table 7 below, and the viable cell count and crude saponin content thereof were measured and shown in Table 7 below.

Culture solution Example 16 Example 17 Example 18 Example 19 Liquid volume (mL) 1,000 1,000 5,000 1,000 Number of living cells (cfu / mL) 2.61E + 09 2.61E + 09 2.61E + 09 2.61E + 09 Crude saponin content (%) 0.62 0.62 0.62 0.62
Centrifugation RPM 2,000 1,000 3,000 2,000 Time (minutes) 10 10 - - system Batch type Batch type Continuous Continuous
Separation liquid Separated liquid volume (mL) 917 945 4385 4451 Number of living cells (cfu / mL) 2.10E + 09 2.51E + 09 2.25E + 09 2.31E + 09 Viable cell viability (%) 73.8 90.9 75.6 78.8 Crude saponin content (%) 0.62 0.61 0.62 0.62 Crude saponin remaining ratio (%) 91.7 93 87.7 89

Example  20 to 23

The centrifugation conditions of the culture solution prepared in Example 13 and Example 14 were changed to remove the precipitate and prepare a separation liquid. The centrifugation conditions were changed as shown in Table 8, and the viable cell count and crude saponin content were measured, and the results are shown in Table 8 below.

Culture solution Example 20 Example 21 Example 22 Example 23 Liquid volume (mL) 20,000 20,000 20,000 20,000 Number of living cells (cfu / mL) 3.20E + 09 3.20E + 09 1.98E + 09 1.98E + 09 Crude saponin content (%) 0.72 0.72 0.65 0.65
Centrifugation RPM 3,000 2,000 4,000 1,500 Time (minutes) - - - - system Continuous Continuous Continuous Continuous
Separation liquid Separated liquid volume (mL) 16,310 14,820 17,850 11,950 Number of living cells (cfu / mL) 2.60E + 09 2.90E + 09 1.35E + 09 1.70E + 09 Viable cell viability (%) 66.3 67.2 60.9 51.3 Crude saponin content (%) 0.68 0.67 0.60 0.61 Crude saponin remaining ratio (%) 77.0 68.9 82.4 56.1

Example  24 to 26

The centrifugation conditions of the culture solution prepared in Example 15 were changed to remove the precipitate, and a separation liquid was prepared. The centrifugation conditions were changed as shown in Table 9, and the viable cell count and crude saponin content were measured and shown in Table 9 below.

Culture solution Example 24 Example 25 Example 26 Liquid volume (mL) 20,000 20,000 20,000 Number of living cells (cfu / mL) 2.40E + 09 2.40E + 09 2.40E + 09 Crude saponin content (%) 0.66 0.66 0.66
Centrifugation RPM 3,000 2,000 1,800 Time (minutes) - - - system Continuous Continuous Continuous
Separation liquid Separated liquid volume (mL) 16,874 14,568 13,256 Number of living cells (cfu / mL) 1.80E + 09 2.12E + 09 2.31E + 09 Viable cell viability (%) 63.3 64.3 63.8 Crude saponin content (%) 0.63 0.63 0.63 Crude saponin remaining ratio (%) 80.6 69.5 63.3

Example  27 to 31 and Comparative Example  One

The liquid prothyroscopic preparations of Examples 27 to 31 and Comparative Example 1 were prepared by mixing the separated solution, organic acid and gum compounds prepared in Example 24 according to the mixing ratios shown in Table 10 below.

The stability of the liquid probiotic composition prepared at the time of 0 week, 2 weeks, and 4 weeks at a temperature of 25 ± 1 ° C. and a relative humidity of 75 ± 5% was evaluated and the properties such as the occurrence of precipitates were evaluated.

Example 27 Example 28 Example 29 Example 30 Example 31 Comparative Example 1 Organic acid Citric acid 0.20 0.20 0.40 0.50 0.50 0 Sword (GUM) Xanthan gum 0.10 0.20 0.10 0.10 0.20 0 Culture solution Example 24 99.70 99.60 99.50 99.40 99.30 100 0 weeks
Viable cell count 1.80E + 09 1.80E + 09 1.80E + 09 1.80E + 09 1.80E + 09 1.80E + 09 pH 3.59 3.62 3.25 3.05 3.03 5.92 precipitate Yes / No (X) X X X X X ○ stability Yes / No (X) ○ ○ ○ ○ ○ X 2 weeks
Viable cell count 1.75E + 09 1.71E + 09 1.72E + 09 1.70E + 09 1.76E + 09 1.71E + 09 pH 3.65 3.59 3.32 3.06 3.08 6.12 precipitate Yes / No (X) X X X X X ○ stability Yes / No (X) ○ ○ ○ ○ ○ X 4 weeks
Viable cell count 1.76E + 09 1.72E + 09 1.73E + 09 1.76E + 09 1.75E + 09 Pollution pH 3.58 3.59 3.28 3.07 3.04 - precipitate Yes / No (X) X X X X X ○ stability Yes / No (X) ○ ○ ○ ○ ○ X

Example  32 to 36 and Comparative Example  2

The liquid preparations of Examples 32 to 36 and Comparative Example 2 were prepared by mixing the separated solution, organic acid and gum compounds prepared in Example 24 according to the mixing ratios shown in Table 11 below.

The stability of the liquid type prophylactic composition thus prepared at the time of 0 week, 2 weeks and 4 weeks at a temperature of 40 ± 1 ° C and a relative humidity of 75 ± 5% was evaluated and the properties such as occurrence of precipitates were evaluated.

Example 32 Example 33 Example 34 Example 35 Example 36 Comparative Example 2 Organic acid Citric acid 0.20 0.20 0.40 0.50 0.50 0 Sword (GUM) Xanthan gum 0.10 0.20 0.10 0.10 0.20 0 Culture solution Example 24 99.70 99.60 99.50 99.40 99.30 100 0 weeks
Viable cell count 1.80E + 09 1.80E + 09 1.80E + 09 1.80E + 09 1.80E + 09 1.80E + 09 pH 3.59 3.62 3.25 3.05 3.03 5.92 precipitate Yes / No (X) X X X X X ○ stability Yes / No (X) ○ ○ ○ ○ ○ X 2 weeks
Viable cell count 1.78E + 09 1.75E + 09 1.76E + 09 1.71E + 09 1.75E + 09 Pollution pH 3.65 3.59 3.32 3.06 3.08 6.12 precipitate Yes / No (X) X X X X X ○ stability Yes / No (X) ○ ○ ○ ○ ○ X 4 weeks
Viable cell count 1.71E + 09 1.69E + 09 1.73E + 09 1.71E + 09 1.75E + 09 Pollution pH 3.58 3.59 3.28 3.07 3.04 - precipitate Yes / No (X) X X X X X ○ stability Yes / No (X) ○ ○ ○ ○ ○ X

<Efficacy evaluation>

end. Example  Evaluation of efficacy of culture broth 1

1) Evaluation of ginsenoside content

The test samples were prepared by dividing the culture solution of Example 1 by the ginsenoside test method in the "Standards and Specifications of Health Functional Foods" described in the Health Functional Food Cycle, before and after culturing for 48 hours, and measuring the concentrations of ginsenosides Rb1, The contents of Rg1 and Rg3 were measured. Specifically, sufficient ginsenoside components were extracted from each sample using distilled water and an organic solvent, and analyzed quantitatively at a maximum absorption wavelength of 203 nm using a liquid chromatograph / ultraviolet absorbance detector. The standard solution, the preparation conditions of the test solution and the conditions of the high-performance liquid chromatograph are shown below.

- Preparation of standard solution: 10 mg of ginsenosides Rb1, Rg1 and Rg3, respectively, are placed in a 10 mL volumetric flask. Fully dissolve in methanol and fill the methanol to the mark (1 mg / mL). It was diluted appropriately and made into a standard solution.

- Preparation of test solution: Each sample was filtered (0.45 μm) to prepare test solution.

- Fast chromatogram conditions: 20 μl injection volume, 203 nm detector wavelength, 30 ° C column temperature, mobile phase A distilled water, mobile phase B acetonitrile, flow rate 1.0 mL / min, mobile phase conditions (Table 11).

Time (minutes) Mobile phase A solution (%) Mobile phase B solution (%) Time (minutes) Mobile phase A solution (%) Mobile phase B solution (%) 0 80 20 35 50 50 5 80 20 75 15 85 20 77 23 77 15 85 25 70 30 80 80 20 30 60 40 100 80 20

The measured values (measured values for the respective Rb1, Rg1, and Rg3 samples) were corrected by substituting the following equation 1, and the results are shown in Table 12 (unit: 쨉 g / mL).

[Formula 1]

Content of ginsenosides (Rb1, Rg1, Rg3) (μg / g) = C X (a X b) / S

Wherein C is the concentration of individual ginsenoside (쨉 g / mL) in the test solution, a is the total amount (mL) of the test solution, b is the dilution factor, and S is the sample weight (g).

Ginsenoside type Pre-culture sample After 48 hours of incubation, Rate of increase / decrease (%) Rb1 8.98 25.20 280.6 Rg1 1.34 3.84 286.6 Rg3 15.65 49.90 318.8

2) DPPH  Radical Scatters (Antioxidant)

As the positive control, 1 mg / mL, 0.1 mg / mL, and 0.01 mg / mL of ascorbic acid were used.

0.0039 g of DPPH was dissolved in 100 mL of methanol to prepare DPPH 0.1 mM. To the 3.8 mL of DPPH methanol solution, 0.2 mL of the sample was added, vortexed for about 1 minute, and then reacted at room temperature for 30 minutes. The absorbance was then measured at 517 nm.

Positive control was carried out in the same manner as the sample. Absorption of DPPH methanol solution was measured as a negative control. % discolouration was calculated by [1- (sample O.D value / negative control O.D value)] × 100, and the results are shown in Table 14 (unit:% Discolouration).

  The results of% Discolouration showed 13.7%, 13.0% at 24 hours and 10.1% at 48 hours in the culture time 0 h, In the fermentation broth containing red ginseng, the culture time was 13.7% at 0 h, 23.9% at 24 h, and 28.7% at 48 h. As a result, the antioxidant capacity was increased 48 hours after fermentation.

Culture time (hours) Non-strain-added group Strain-added group 0 13.7 13.7 24 13.0 23.9 48 10.1 28.7

I. SD RAT general growth characteristics analysis

- Preparation of test animals: SD rats were fed in the rats, and the animals were adapted for 2 weeks while being fed free of general solid feed and water.

1) Analysis of SD Rat growth characteristics

Three male Sprague Dawley rats of 3 weeks old were divided into three groups: normal control (Negative control), positive control (0.1% of red ginseng solution), 0.1% of liquid fermented red ginseng solution of Example 1, 1% administration group of liquid ginseng roots fermented product of Example 1 of the present invention and 5% administration group of liquid red ginseng fermentation product of Example 1 of the present invention were divided into 6 rats / group for drinking water for 2 weeks, And body weight, etc. The results are shown in Table 15. &lt; tb &gt; &lt; TABLE &gt;

Weight (g) Control group Normal group 0.1% treatment group 1.0% treated group 5.0% treated group Before initiation of treatment 103.53 + - 3.90 98.72 + - 8.05 94.91 + - 4.11 105.30 ± 2.39 93.69 ± 5.90 After the end of administration 197.35 ± 15.84 185.79 ± 9.34 182.27 ± 13.58 208.59 ± 13.03 168.59 + - 10.41 Gross weight gain 93.82 + - 11.68 87.07 8.08 87.36 +/- 12.45 103.29 ± 13.53 74.89 ± 10.54 Total feed intake 248.92 + - 2.57 239.12 + - 2.12 233.38 ± 12.45 262.82 ± 3.16 223.21 + - 2.35 Feed efficiency 38% 36% 37% 39% 34%

2) Evaluation of long term weight change of SD Rat

After completion of the analysis of the growth characteristics, the animals were euthanized at the end of the experiment, and the spleen, liver, and thymus were excised and their weights were measured. The change of each organ organs was visually observed by the scale of toxicity reduction and the change of weight of each immune organ was observed by percentage (%) of body weight. The results are shown in Table 16 below.

long time
(g / 100 g bw) Control group Normal group 0.1% treatment group 1.0% treated group 5.0% treated group P value spleen 0.34 + 0.04 0.31 + 0.02 0.35 + 0.03 0.33 + 0.02 0.32 ± 0.03 0.45 liver 4.91 ± 0.53 4.67 ± 0.37 4.21 + 0.09 4.44 ± 0.48 4.32 0.31 0.21 Thymus 1.45 ± 0.07 1.34 ± 0.06 1.17 ± 0.09 1.32 ± 0.05 1.25 + 0.07 0.72

All. Pigeon  Analysis of growth characteristics

One) Pigeon  Weight change

Body weights were determined by randomly weighing 10 rats in each group at the beginning of the experiment (0 day) and the end of the experiment (28 days). The weight of the experiment was calculated by subtracting the weight of the experiment start date (0 day) Respectively. 100 weekly 3 - yr crossbreeding (Landrace × Yorkshire × Duroc) finishing pigs were divided into control and experimental groups (T1). In the experimental group, the liquid probiotics composition of Example 1, which was fermented with Bacillus subtilis HD 9098 through an automatic feeder, was mixed with 1% of a general feed. The control group fed only normal diet. Water and feed were measured at baseline physiological changes, while free feeding. The results are shown in Table 17 below.

Control group Experimental group Before the start of feeding (Kg) 8.16 ± 0.97 7.14 ± 0.69 After feeding (Kg) 2.77 ± 18.71 17.70 ± 3.15 Weight change (Kg) 10.56 0.67 Weight gain (%) 1290 152

2 ) Hematology Appearance Rating (Blood Biochemical parameter)

Blood samples were collected from 10 pigs through the jugular vein randomly at the beginning of the experiment (day 0) and end of the experiment (day 28). Half of the collected blood was placed in a BD Vacutainer EDTA tube and refrigerated at 4 ° C. Half of the remaining blood was placed in a BD Vacutainer SST tube and coagulated at room temperature. The blood was centrifuged at 2500 rpm for 15 minutes, Lt; / RTI &gt;

(ALT), aspartate transaminase (AST), total cholesterol (TC) and triglyceride (TG) contents were measured by a biochemical automatic analyzer (Hitachi-747, Hitachi Medical, Co., Ltd. Japan).

- Aspartate aminotransferase (AST) was analyzed by IFCC method. Reagent 1 was prepared by mixing 100 mmol / L of TRIS buffer, 300 mmol / L of L-aspartate, 900 U / L of LPH, 600 U / L of MDH and 0.4 mmol / L of NADH. Reagent 2 contained 60 mmol / L of a-oxoglutarate, 0.9 mmol / L. 100 μl of Reagent 1 was added to 100 μl of sample, and the mixture was reacted for 5 minutes. Reagent 2 was mixed with 250 μl for 1 minute, and the absorbance was measured at 340 nm.

- ALT (Alanine Aminotransferase) was analyzed by IFCC method. Reagent 1 was prepared by mixing TRIS buffer 150 mmol / L, L-alanine 750 mmol / L, LDH 1200 U / L and NADH 0.4 mmol / L. Reagent 2 was prepared by mixing 90 mmol / L of a-oxoglutarate and 0.9 mmol / L of NADH made. 100 μl of Reagent 1 was added to 100 μl of the sample, and the mixture was reacted for 5 minutes. Reagent 2 was mixed with 250 μl of the mixture for 1 minute, and the absorbance was measured at 340 nm.

- Albumin was analyzed by Bromcresol Green method. Reagent was prepared by mixing 30 mmol / L citrate buffer, 0.26 mmol / L bromocresol green and 1.5 g / L surfactant. Reagent 1000 uL was mixed with 10 uL of the sample and incubated at 37 ° C for 5 min. The absorbance was measured at 578 nm.

- Glucose was analyzed by GOD-POD method. Reagent 1 was prepared by mixing 100 mmol / L of phosphate buffer, 4700 U / L of ascorbate oxidase and 4000 U / L of glucose oxidase. Reagent 2 contained 100 mmol / L of phosphate buffer, 6700 U / L of peroxidase, 0.7 mmol / L of 4-aminoantipyrine, hydroxybenzoic acid sodium (1.3 mmol / L). Reagent1 240 μL of the sample was mixed with 3 μL of the sample, reacted at 37 ° C. for 5 minutes, reacted with Reagent 2 (60 μL) for 5 minutes, and the absorbance was measured at 510 nm.

The measurement results are shown in Table 18.

Control group Experimental group AST
(U / L) Before the start of class 77.10 ± 17.93 75.70 ± 24.33 After the class ends 65.44 ± 19.38 66.44 + 19.41 Variation 11. 66 9.26 ALT
(U / L) Before the start of class 45.20 + - 7.45 46.50 ± 9.01 After the class ends 59.67 ± 14.68 48.67 ± 8.73 Variation 14.47 2.17 Albumin
(g / dL) Before the start of class 4.40 ± 0.23 4.19 ± 0.32 After the class ends 3.68 ± 0.16 3.21 ± 0.25 Variation 0.72 0.98 Glucose
(mg / dL) Before the start of class 78.40 ± 19.84 92.60 + - 16.71 After the class ends 113.89 + 14.18 115.89 ± 23.52 Variation 35.49 23.29

3) Evaluation of cholesterol analysis

- Total cholesterol was analyzed by the CHOD-POD method. Reagent was prepared by mixing phosphate buffer 100 mmol / L, phenol 5 mmol / L, 4-aminoantipyrine 0.3 mmol / L, cholesterol esterase 150 KU / L, cholesterol oxidase 100 KU / L and peroxidase 5 KU / L. 10 μl of sample was added to 1000 μl of Reagent, and incubated at 37 ° C for 10 minutes. The absorbance was measured at 510 nm.

- Triglycerides were analyzed by GPO-POD method. Phosphate buffer 50 mmol / L, 4 -cholrophenol 5 mmol / L, ATP 2 mmol / L, Mg 2 + 4.5 mmol / L, glycerokinase 0.4 U / L, peroxidase 0.5 U / mL, lipoprotein lipase 1.3 U / mL, 4- aminoantipyrine 0.25 mmol / L, and glycerol-3-phosphate-oxidase 1.5 U / mL. Reagent 1000 μl was mixed with 10 μl of sample, and reacted at 37 ° C for 10 minutes, and the absorbance was measured at 510 nm.

- HDL-Cholesterol was analyzed by Direct method. Reagent 1 was prepared by mixing 100 mmol / L of good's buffer, 600 U / L of cholesterol esterase, 380 U / L of cholesterol oxidase, 600 KU / L of catalase and 0.42 mmol / L of HDAOS. Reagent 2 contained 100 mmol / amniantipyrine 1.0 mmol / L, peroxidase 2.8 U / mL, and surfactant 2%. Reagent 1 900 μl of the sample was mixed with 12 μL of the reaction mixture, which was then reacted at 37 ° C. for 5 minutes. Reagent 2 was mixed with 300 μL of the reaction mixture at 37 ° C. for 5 minutes, and the absorbance was measured at 600 nm.

- LDL-Cholesterol was analyzed by Direct method. Reagent 1 was prepared by mixing 50 mmol / L of good's buffer, 600 U / L of cholesterol exterase, 500 U / L of cholesterol oxidase, 600 KU / L of catalase and 2 mmol / L of TOOS. Reagent 2 good's buffer 50 mmol / L, 4-aminoantipyrine 4 mmol / L, and peroxidase 4 U / mL. Reagent 1 was mixed with 900 μL of sample 12 μL and reacted at 37 ° C. for 5 minutes. Reagent 300 μL was added to the reaction mixture at 37 ° C. for 5 minutes, and the absorbance was measured at 600 nm.

The measurement results are shown in Table 19.

Control group Experimental group Total Cholesterol
(mg / dL) Before the start of class 183.10 + - 41.23 173.20 ± 38.74 After the class ends 81.00 ± 10.93 82.56 + - 7.84 Variation 102.1 90.64 Triglyceride
(mg / dL) Before the start of class 125.30 ± 49.85 81.50 ± 39.72 After the class ends 46.67 ± 12.44 71.89 ± 11.14 Variation -77.45 9.61 HDL-Cholesterol
(mg / dL) Before the start of class 51.42 + - 6.54 47.95 ± 10.79 After the class ends 31.58 + - 4.91 32.36 + - 2.42 Variation -19.84 -15.59 LDL-Cholesterol
(mg / dL) Before the start of class 121.17 ± 39.72 117.63 ± 36.33 After the class ends 48.04 + - 8.79 46.66 + - 6.66 Variation 73.13 70.97

4) Immunoglobulin  evaluation

IgG, IgM and IgA were measured by immunocytochemistry at Immunoglobulin (Komabiotech) at -70 ℃. Serum was diluted 20,000 times with Assay diluent (1% BSA in PBS) and analyzed using Pig IgG ELISA kit, Pig IgM ELISA kit and Pig IgA ELISA kit. After pre-coated 96-well ELISA microplate was washed with wash buffer (PBST), 100 μL of standard and sample were added in duplicate and incubated at room temperature for 1 hour. After washing, 100 μL of HRP conjugated antigen-affinity purified goat anti-Pig IgG (or IgM, IgA) was diluted 20,000 times in each well and incubated at room temperature for 1 hour. After adding 100 μL of color development reagent (TMB solution) to each well, incubate at room temperature for about 10 minutes, add 100 μL of stop solution (2M H2SO4) The OD value of the standard was used as a standard curve and the OD value of the sample was substituted to obtain the immunoglobulin value of each sample.

Control group Experimental group IgG
(mg / mL) Before the start of class 5.76 ± 1.08 5.35 ± 1.19 After the class ends 7.07 + 1.05 7.44 0.95 Variation 1.36 2.09 IgM
(mg / mL) Before the start of class 5.76 ± 1.08 1.11 ± 0.26 After the class ends 7.07 + 1.05 3.50 + - 0.85 Variation 2.01 2.39 IgA
(mg / mL) Before the start of class 0.02 0.06 0.19 ± 0.05 After the class ends 0.41 + 0.11 0.49 + - 0.10 Variation 0.21 0.30

5) Cytokine (TNF-a, IL-1b)  concentration

Cytokine was assayed for TNF-a, IL-1b, and IL-6 by commissioning a sera stored at -70 ° C to Komabiotech. Serum samples were analyzed by Milliplex® MAP porcine cytokine / chemokine magnetic bead panel kit 96-well plate. Assay buffer was added to each well of a 96-well plate and incubated at room temperature for 10 minutes on a plate shaker. After removal of the assay buffer, 25 μL of each standard was added to each well, and 25 μL of the assay buffer was added to each sample well. Then, 25 μL of serum matrix was added to each well. Samples were added to each well at 25 μL and premixed beads (TNF-a, IL-1b, and IL-6) were added to all wells. The 96-well plate was sealed and incubated at 4 ° C on a plate shaker. After washing, 50 μl of detection antibodies were added to each well and incubated on a plate shaker at room temperature for 2 hours. After that, streptavidin-phycoerythrin was added to each well in an amount of 50 μL and incubated on a plate shaker for 30 minutes at room temperature. After washing, 100 μL of sheath fluid was added to all wells, and the reaction was carried out on a plate shaker for 5 minutes and analyzed with Luminex® 200 ™. The concentration of each cytokine in the sample was determined from the results of the analyzed standard.

Control group Experimental group TNF-a
(ng / mL) Before the start of class 0.96 + 0.19 0.84 + 0.24 After the class ends 1.70 + - 0.24 0.57 ± 0.12 Variation 1.46 -0.27 IL-1?
(ng / mL) Before the start of class 7.23 + 1.47 6.83 ± 1.19 After the class ends 7.99 ± 1.19 6.97 + 1.35 Variation 0.76 0.14 IL-6
(ng / mL) Before the start of class 2.71 0.37 2.85 0.47 After the class ends 3.57 ± 0.29 3.15 ± 0.36 Variation 0.76 0.30

6) Evaluation of cholera antibody changes

Serum of porcine cholera antibody stored at -70 ° C was measured by a co-worker Permission for inspection. Porcine cholera antibody was analyzed using a classical swine fever virus (CSFV) antibody ELISA kit. Positive / negative controls and samples diluted 20-fold in CSFV gp55 coated plates were added to each well in an amount of 100 μL and incubated at 37 ° C for 1 hour. After washing, 100 μL of HRPO Anti-Swine IgG Conjugate was added to each well and incubated at 37 ° C for 1 hour. After washing, 100 uL of each coloring agent (ABTS Substrate) was added to each well. After reacting at room temperature for 10 minutes, stop solution was added to each well. The absorbance was then measured at 405 nm with an ELISA reader. S / P = (Sample mean OD value-Negative control mean OD value) was calculated by subtracting the average OD value of the negative control from the positive control value of the positive control group to obtain Corrected Positive Control (CPC) ) / CPC, and the result was judged as positive when the value was 0.14 or more, and negative when it was less than 0.14.

Control group Experimental group CSFV ab
(S / P) Before the start of class 0.75 + 0.07 0.72 + 0.13 After the class ends 0.31 ± 0.05 0.30 ± 0.06 Variation 0.44 0.41

7) Piglet Lichen  change

 During the pond specification test, the fungus was collected for 5 animals in each of the control group and the test group three times after the start of the test, after 2 weeks, and after 4 weeks. Measured microorganisms were aerobic microbial bacteria, coliform bacteria, intestinal bacteria, diluted 1 g of each feces in sterilized diluted water, and diluted appropriately in each experimental group by decidual dilution method. - The medium was aerobic count plates, E. coli / Coli form count plates and Enterobacteriaceae count plates among 3M petri films. Colony colonies were counted after culturing at 36 ± 1 ℃ for 48 hours.

Control group Aerobic Count Coli form Enterobacteriacaea 0 6.44E + 07 4.84E + 04 6.50E + 04 2 9.24E + 07 1.07E + 05 1.77E + 06 4 5.50E + 08 2.50E + 06 4.16E + 06 Experimental group Aerobic Count Coli form Enterobacteriacaea 0 6.44E + 07 4.84E + 04 6.50E + 04 2 4.04E + 07 2.61E + 04 2.11E + 05 4 3.56E + 08 5.02E + 05 2.12E + 06

As shown in the above Table 23, the increase of the increase in the coliform group was observed at 2 weeks and 4 weeks after the start of the test as compared with that of the control group. After the week, it showed similar tendency to the control group.

Korea Research Institute of Bioscience and Biotechnology KCTC18456P 20160308

Claims (5)

Bacillus subtilis HD 9098 (bacillus subtilis HD 9098, KCTC18456P), which has antibacterial activity against pathogenic bacteria, microbial antibiotic activity against pathogens, and red ginseng rooting effect and has an effect of increasing crude saponin content and ginsenoside liberation rate during fermentation of red ginseng, Strain.
A fermentation broth preparation step of fermenting red ginseng roots using a culture solution of Bacillus subtilis HD 9098 (bacillus subtilis HD 9098, KCTC 18456P);
A separation liquid preparation step of removing the solid content from the fermentation culture liquid; And
A step of preparing a biocide by adding at least one of organic acid and gum compounds to the separated solution and liquefying it; &Lt; / RTI &gt;
3. The method of claim 2,
In the fermentation broth preparation step, Bacillus subtilis HD 9098 (Bacillus subtilis HD 9098, KCTC 18456P) was inoculated into a medium containing 0.01% to 50% by weight of red ginseng roots on the basis of a 5 L to 5000 L fermenter, , And subjecting the culture broth to liquid fermentation at a temperature of 15 ° C to 45 ° C for 6 to 120 hours by adjusting the amount of aerosolization.
3. The method of claim 2,
The step of producing the liquid probiotics is performed by adding 0.01 to 50.0% by weight of organic acid, 0.001 to 10.0% by weight of gum compounds and 0.01 to 80% by weight of distilled water to 10.0 to 99.90% , &Lt; / RTI &gt;
Wherein the organic acid comprises at least one of citric acid and acetic acid and the gum compounds include at least one of xanthan gum, guar gum and gellan gum &Lt; / RTI &gt;
A prophylactic or therapeutic composition according to any one of claims 2 to 4,
The probiotic composition may be selected from the group consisting of Bacillus subtilis HD 9098, KCTC 18456P strain, cellulase, amylase, lipase, protease, pedctinase, crude saponin, Ginsenoside Rb1, ginsenoside Rg1, and ginsenoside Rg3,
Wherein the viable composition has a viable cell viability of 90% or more and a crude saponin residual ratio of 95% or more after an accelerated condition test in which the biocidal composition is allowed to stand for 4 weeks at a temperature of 40 ± 1 ° C and a relative humidity of 75 ± 5%.

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