KR101943862B1 - Method of concentrating killed-lactic acid bacteria using membrane filter - Google Patents

Abstract

The present invention relates to a method for concentrating a microbial strain of lactic acid bacteria, which comprises passing a culture broth of a lactic acid bacterium containing a medium containing microbial strains of lactic acid bacteria through a membrane filter.

Description

METHOD OF CONCENTRATING KILLED-LACTIC ACID BACTERIA USING MEMBRANE FILTER "

The present invention relates to a method for concentrating a microbial strain of lactic acid bacteria, which comprises passing a culture broth of a lactic acid bacterium containing a medium containing microbial strains of lactic acid bacteria through a membrane filter.

The definition of probiotics, as designated by the World Health Organization (WHO), refers to live microorganisms that, when applied in an appropriate amount, can provide health benefits to the host. host). However, recently, the commercialization of probiotics of lactic acid bacteria is rapidly expanding, and the bacteria are also used as probiotics in the probiotic market.

As probiotics, probiotics have a variety of advantages over probiotics, and they are already commercially available in Japan and the United States. The probiotics can be used more stable than probiotics, so they have a wide range of industrial applications and are easy to handle in the distribution process. They are added to the general food to enhance the functionality of the food while showing the same effect as the probiotics in the immune control function Recently, the market has increased significantly.

As a conventional technique for producing such dead bacteria, there is a batch-type culture method. The batch-type culture method generally includes culturing a culture medium for growth of lactic acid bacteria and lactic acid bacteria in a large-capacity culture medium, and then heating and culturing the cultured lactic acid bacteria (Korean Patent Laid-Open Publication No. 10-2012-0047792) .

However, dead cells from such batch culture have the following disadvantages.

First, since impurities generated during the cultivation of lactic acid bacteria can not be filtered quickly, a separate step for washing the lactic acid bacteria is required, and the lactic acid bacteria culturing time may take a relatively long time. Secondly, it is not possible to selectively remove impurities from the culture medium itself or impurities produced by the cultured lactic acid bacteria in the culture medium containing the cultured lactic acid bacteria.

Therefore, it is not possible to selectively concentrate the lactic acid bacteria in the culture medium containing the cultured lactic acid bacteria, and thus it is impossible to produce lactic acid bacteria having high concentration and high quality. Particularly, the lactic acid bacteria produced by the batch culture grows as the lactic acid bacteria accumulate in the heat treatment process. Accordingly, when the lactic acid bacteria prepared by the batch culture are added to the food or beverage, they precipitate and cause a suspension phenomenon.

In order to solve the disadvantages of the prior art as described above, the present invention provides a method for concentrating a microbial strain of lactic acid bacteria, comprising passing a culture broth of a lactic acid bacterium containing a microbial strain containing microbes of Mycobacterium tuberculosis through a membrane filter.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

According to a first aspect of the present invention, there is provided a method for concentrating a bacteria of a lactic acid bacteria, which comprises passing a culture broth of a lactic acid bacterium containing a medium containing killed bacteria of a lactic acid bacteria to a membrane filter.

The second aspect of the present invention provides a method for producing microbes of lactic acid bacteria, which comprises the above-described method for concentrating microbes.

The third aspect of the present invention provides a killed microbial strain of lactic acid bacteria produced by the above production method.

A fourth aspect of the present invention provides an additive comprising the above dead cells.

According to embodiments of the present application, the lactic acid bacteria bacteria can be concentrated and dispersed by passing a culture solution of the lactic acid bacteria containing the medium containing the lactic acid bacteria dead bacteria through a membrane filter.

According to the embodiments of the present invention, the culture broth of lactic acid bacteria containing the culture medium containing the lactic acid bacteria dead cells is passed through the membrane filter to disperse the aggregated lumps of the lactic acid bacteria in the process of desizing the lactic acid bacteria. Further, since the impurities contained in the medium are filtered, it is also possible to wash out the dead bacteria of the above-mentioned lactic acid bacteria.

According to the embodiments of the present invention, impurities such as proteins, carbohydrates, and lactic acid bacterial rumble other than the lactic acid bacteria can be filtered using the membrane filter, thereby providing a strain of high concentration of lactic acid bacteria.

Furthermore, according to the embodiments of the present invention, since the washing process of the lactic acid bacteria can be simplified or the washing process may not be required, the production time of the lactic acid bacteria and / or dead bacteria can be shortened.

According to embodiments of the present invention, a culture medium containing lactic acid bacteria dead cells is passed through the membrane filter to disperse and concentrate the agglomerated mass of lactic acid bacteria, thereby providing a high-quality lactic acid bacteria dead bacteria. When such a high-quality lactic acid bacteria-containing dead bacteria is added to a food, a functional food or a feed, it does not cause a precipitation phenomenon, and thus can exhibit excellent flowability and storage effect.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic view of a method for producing microbes of lactic acid bacteria comprising a method for concentrating microbes of mycobacterial strains according to one embodiment of the present invention. Fig.
FIG. 2 is a schematic diagram comparing the structures of a conventional bioreactor and a conventional batch-type lactic acid bacteria incubator according to an embodiment of the present invention. FIG.
FIG. 3 is a graph showing productivity, growth rate, and concentration of cultured and concentrated lactic acid bacteria and lactic acid bacteria in accordance with one embodiment of the present invention and lactic acid bacteria cultured according to Comparative Example 1. FIG.
FIG. 4 is a photograph (SEM, X10,000) of a microphotograph of a lyophilized lactic acid bacteria and a lactic acid bacteria prepared according to Comparative Example 1, according to an embodiment of the present invention.
FIG. 5A is a photograph (magnification, X1, 000) of the sizes of cultured and concentrated lactic acid bacteria and lactic acid bacteria grown in accordance with one embodiment of the present invention and lactic acid bacteria cultured according to Comparative Example 1. FIG.
FIG. 5B is a photograph (magnified with an optical microscope, X1, 000) of the size of bacteria killed in accordance with one embodiment of the present invention and the bacteria killed in accordance with Comparative Example 1. FIG.
FIG. 6 is a graph comparing the size distributions of lactic acid bacteria cultured and concentrated according to one embodiment of the present invention and lactic acid bacteria bacteria cultured according to Comparative Example 1. FIG.
FIG. 7 is a graph comparing the size distributions of dead bacteria-killed lactic acid bacteria and lactic acid bacteria produced according to Comparative Example 1 according to one embodiment of the present invention.
8 is a photograph showing the size of each lactic acid bacterium immediately after the cultivation and concentration of the lactic acid bacterium and immediately after the lysis of the lactic acid bacterium and immediately after the concentration and washing of the bacteria of the lactic acid bacteria in the production method according to one embodiment of the present invention , X1, 000).
FIG. 9 is a graph comparing changes in size before and after passage of the bacteria of the lactic acid bacteria produced according to one embodiment of the present invention into the second membrane filter.
FIG. 10 is a photograph comparing the properties, that is, the chromaticity, of the raw material of the killed microbes of the lactic acid bacteria produced according to one embodiment of the present invention and the comparative example.
Fig. 11 is a comparison of the result of suspending the raw materials of dead bacteria of the lactic acid bacteria produced according to one embodiment of the present invention and the comparative example in distilled water.
FIG. 12 is a graph showing the immunological activity of a lactic acid bacterium according to an embodiment of the present invention.
FIG. 13 is a graph showing the immunological activity of killed bacteria of the lactic acid bacteria according to one embodiment of the present invention. The RAW 264.7 cell line, which is a type of macrophage, is used to generate cytotoxicity (LDH) and nitric oxide . Fig.
FIG. 14 is a graph showing the immunological activity of mycobacterium tuberculosis according to one embodiment of the present invention. FIG. 14 is a graph showing cytotoxicity (LDH) and TNF-.alpha., INF-.gamma., IL- 4 is a graph showing the degree of production.
FIG. 15 is a photograph and a graph showing the anti-obesity effect of the killed bacteria of the lactic acid bacteria according to one embodiment of the present invention, showing a decrease in fat accumulation in the fat cells inversely proportional to the increase in the concentration of treated bacteria And the graph.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In the drawings, the same reference numbers are used throughout the specification to refer to the same or like parts.

Throughout this specification, when a part is referred to as being "connected" to another part, it is not limited to a case where it is "directly connected" but also includes the case where it is "electrically connected" do.

Throughout this specification, when a member is " on " another member, it includes not only when the member is in contact with the other member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as " including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise. The terms " about ", " substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) " or " step " used to the extent that it is used throughout the specification does not mean " step for.

Throughout this specification, the term " combination (s) thereof " included in the expression of the machine form means a mixture or combination of one or more elements selected from the group consisting of the constituents described in the expression of the form of a marker, Quot; means at least one selected from the group consisting of the above-mentioned elements.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Throughout this specification, the description of a " bioreactor " means " the entire system as a whole capable of producing microorganisms such as lactic acid bacteria ".

Throughout the present specification, the term "culture medium" means "culture medium containing lactic acid bacteria", and the term "culture medium inoculated with lactic acid germs", "culture medium containing lactic acid bacteria", "cultured and concentrated using a membrane filter" Medium containing lactic acid bacteria, " and " medium containing concentrated, dispersed and washed lactic acid bacteria using a membrane filter ".

In the entire specification of the present application, the term " size of lactic acid bacteria " means the size of one or one individual of lactic acid bacteria, and the specific measuring method differs depending on the type and form of the lactic acid bacteria. When the lactic acid bacterium is a bacterium, it does not mean the length of the bacterium but means the thickness or width of the bacterium. When the lactic acid bacterium is a bacterium, the " size of the lactic acid bacterium " means the diameter of the bacterium. Bifidobacterium lactic acid bacteria and other lactic acid bacteria means the size measured on the basis of the size commonly used by ordinary technicians.

According to a first aspect of the present invention, there is provided a method for concentrating a bacteria of a lactic acid bacteria, which comprises passing a culture broth of a lactic acid bacterium containing a medium containing killed bacteria of a lactic acid bacteria to a membrane filter.

In one embodiment of the invention, impurities contained in the medium may be filtered through the membrane filter.

In one embodiment of the present invention, the lactic acid bacteria culture solution containing the culture medium containing the lactic acid bacteria dead cells may be passed through a membrane filter so that the bacteria can be washed out from the impurities.

In one embodiment of the present invention, the lactic acid bacteria microbes may be dispersed from the lumps of the lactic acid bacteria by passing the culture broth of the lactic acid bacteria containing the medium containing the lactic acid bacteria dead bacteria through a membrane filter. Here, the mass of the dead bacteria of the lactic acid bacteria may be one in which dead bacteria of the lactic acid bacteria are formed in the process of desizing the lactic acid bacteria.

In one embodiment of the invention, the membrane filter may comprise a hollow fiber membrane filter comprising a pore having a diameter of from 0.01 mu m to 0.1 mu m.

In one embodiment of the invention, the membrane filter may comprise a hollow fiber membrane filter for filtering materials having a molecular weight of 50,000 daltons to 500,000 daltons.

In one embodiment of the invention, the lactic acid bacteria may include those selected from bacilli, streptococci, bifidobacteria, and combinations thereof.

In one embodiment of the invention, the lactic acid bacterium may comprise Lactobacillus plantarum .

The second aspect of the present invention provides a method for producing microbes of lactic acid bacteria, which comprises the above-described method for concentrating microbes.

The third aspect of the present invention provides a killed microbial strain of lactic acid bacteria produced by the above production method.

A fourth aspect of the present invention provides an additive comprising the above dead cells.

In one embodiment of the invention, the additive may include those that may be added to food, functional food or feed.

Hereinafter, embodiments and examples of the present invention will be described in detail. However, the present invention is not limited to these embodiments and examples.

In one embodiment of the present invention, the membrane filter may be contained in a bioreactor, and the bioreactor may include an incubator and a membrane filter.

In one embodiment of the invention, the membrane filter includes a first membrane filter and a second membrane filter, so that a shearing force can be applied to the bacteria of the lactic acid bacteria passing through the first membrane filter and the bacteria of the second passage passing through the second membrane filter .

The concentration method according to one embodiment of the present invention can easily adjust the size of the lactic acid bacteria by applying a shearing force to the lactic acid bacteria live bacteria and the dead bacteria of the lactic acid bacteria, and in particular, the size of the lactic acid bacteria can be controlled small. Accordingly, the lactic acid bacterium produced by the concentration method according to one embodiment of the present invention is not only effectively absorbed into the body but also effectively absorbed into the body to exhibit immunological activity and anti-obesity effect.

Here, the magnitude of the shear force applied to the fluid passing through the membrane filter can be expressed by the equation of Hargen-Poiseuiller of Equation (1).

[Formula 1]

In Equation 1, j represents the shear force of the fluid flowing along the pipe, m represents the viscosity of the fluid, Q represents the flow velocity of the fluid, r represents the radius of the pipe, and L represents the length of the pipe. Also, the flow rate of the fluid during culturing can serve as an important factor controlling the shear force.

In one embodiment of the invention, the bioreactor comprising the incubator and the membrane filter may preferably comprise an incubator, a first membrane filter and a second membrane filter. Here, the bioreactor may include a structure in which the fluid added to the incubator passes through the first membrane filter and the second membrane filter independently, and is then transferred to the incubator. 2 is a schematic view of a bioreactor in which the membrane filter (6) includes both a first membrane filter and a second membrane filter, and after the fluid has passed through the first membrane filter and delivered to the incubator, May be passed through a second membrane filter and delivered to the incubator. In one embodiment of the present invention, the first membrane filter and the second membrane filter may exist in mutually independent spaces, unlike in FIG.

Furthermore, the bioreactor may further include a tank for preparing a culture medium and a membrane filter for preparing a culture medium. Here, the medium prepared in the tank for producing medium can be passed through the membrane filter for preparing the medium again, and then delivered to the tank for producing medium or delivered to the incubator. When the medium having passed through the membrane filter for preparing a medium is transferred to an incubator, the medium can be directly passed through the membrane filter for preparing a medium and delivered to the incubator. Also, as shown in FIG. 2, the medium may be stored in a storage tank (3) before being delivered to the incubator, and then transferred to the incubator.

The lactic acid germ cells can be cultured and concentrated by passing the culture medium inoculated with the lactic acid germ cells in the first membrane filter. The cultured and concentrated cultured lactic acid germs can be concentrated and dispersed by passing the culture medium containing the killed microorganisms of lactic acid bacteria through the second membrane filter.

 In one embodiment of the present invention, the first membrane filter may be a hollow fiber membrane filter including a pore having a diameter that does not allow the lactic acid germ cells to be filtered while filtering out impurities contained in the culture medium inoculated with the lactic acid germ cells. The size of the pore diameter of the first membrane filter may be, for example, 0.1 탆 to 1.0 탆, 0.2 탆 to 1.0 탆, 0.3 탆 to 1.0 탆, 0.4 탆 to 1.0 탆, 0.5 탆 to 1.0 탆, Mu m, 0.1 mu m to 0.8 mu m, 0.1 mu m to 0.7 mu m, 0.1 mu m to 0.6 mu m, or 0.1 mu m to 0.5 mu m. If the size of the pore diameter of the first membrane filter is less than 0.1 탆, extreme fouling phenomenon occurs during the cultivation of the lactic acid bacteria live bacteria, and the flux of the culture liquid is reduced. Accordingly, the concentration efficiency of the lactic acid bacteria may be reduced. If the size of the pore diameter of the first membrane filter is more than 1.0 占 퐉, there is a possibility that the lactic acid bacteria live bacteria may be filtered through the membrane together with the impurities.

In one embodiment of the present invention, the second membrane filter is a hollow fiber membrane including a pore having a diameter large enough to concentrate lactic acid bacteria having a reduced size in the culture and concentration process and / Filter. Wherein the pore diameter of the second membrane filter, for example, 0.01 ㎛ to 0.1 ㎛, 0.02 ㎛ to 0.1 ㎛, 0.03 ㎛ to 0.1 ㎛, 0.04 ㎛ to 0.1 ㎛, 0.05 ㎛ to 0.1 ㎛, 0.01 ㎛ to 0.09 ㎛, it may be 0.01 ㎛ to 0.08 ㎛, 0.01 ㎛ to 0.07 ㎛, 0.01 ㎛ ㎛ to 0.06 or 0.01 to 0.05 ㎛ ㎛. If the size of the pore diameter of the second membrane filter is less than 0.01 mu m , an extreme fouling phenomenon occurs in the process of concentrating the pathogenic bacteria of the lactic acid bacteria, thereby reducing the flux of the culture liquid. Thus, there is a possibility that the concentration efficiency of the above-mentioned lactic acid bacteria dead cells may decrease. If the size of the pore diameter of the second membrane filter is more than 0.1 탆 , there is a possibility that the pathogenic bacteria of the lactic acid bacteria are filtered together with impurities through the membrane.

In one embodiment of the present invention, the first membrane filter and the second membrane filter may each independently include a hollow fiber membrane filter having a radius of 6.0 mm or less. When the radius of the hollow fiber membrane filter is 6.0 mm or less, the shearing force applied to the lactic acid bacteria live bacteria or dead bacteria can be increased according to the formula (1). Accordingly, the size of the lactic acid bacteria live bacteria or dead bacteria can be controlled to be small and the degree of dispersion can be increased.

In one embodiment of the present invention, the second membrane filter may include a hollow fiber membrane filter capable of filtering a material having a molecular weight of 50,000 daltons to 500,000 daltons.

In one embodiment of the invention, the bioreactor may include a pressure gauge and / or a flow meter. Here, the pressure gauge is for managing the fouling phenomenon of the membrane filter, and the flow meter measures the flow rate ( Q ) of the culture liquid passing through the membrane filter in real time to adjust the shearing force applied to the lactic acid bacteria .

In one embodiment of the present invention, the bioreactor may further include a tank for preparing a culture medium and a membrane filter for preparing a culture medium. The medium serves to supply nutrients necessary for the cultivation of lactic acid germ cells, and can be prepared by sterilizing the medium solution. Conventional media are usually sterilized by steam or the like. In the case of sterilization of the steam or the like, impurities carbonated by heat generated by carbohydrates, proteins, and the like are generated and remain in the medium, so that the quality of the lactic acid bacteria powder may deteriorate have.

In one embodiment of the present invention, when the bioreactor includes a tank for preparing a medium and a membrane filter for preparing a medium, the impurities contained in the medium solution are filtered using the membrane filter for preparing the medium, Can be prevented. Accordingly, the manufacturing method according to one embodiment of the present invention can provide a high-quality lactic acid bacterial raw material. Here, the membrane filter for preparing a culture medium may include a hollow fiber membrane filter having a diameter enough to filter the impurities contained in the culture medium solution. The diameter may be 0.1 to 1 占 퐉, but is not limited thereto.

In one embodiment of the invention, the lactic acid bacteria may be selected from the group consisting of bacilli, streptococci, bifidobacteria, and combinations thereof. For example, Lactobacillus plantarum, L. acidophilus, L. reuteri, L. gasseri, L. crispatus, L, rhamnosus, L. casei, L. sakei, L. curvatus, L. shirota, L. reuteri, L. fermentum , L. brevis Bacillus and Lactococcus lactis , Lactococcus lactis subsp . lactis , Streptococcus thermophilus , Enterococcus faecium , Enterococcus facalis , and Bifidobacterium longum , B. lactis , B. lactis subsp . latis, B. there may be mentioned a bipyridinium dogyun of infantis, B.breve, B. aldolescence, and the like. In one embodiment of the invention, if the lactic acid bacteria including L. plantarum (LM1001, Accession No. KCCM 42 959) or L.plantarum (LM1004, KCCM deposit number 43 246), may appear to the effect of the present application more pronounced than .

In one embodiment of the present invention, the lactic acid germ cells inoculated into the culture medium may be cultured through one or more culture steps. For example, the lactobacillus can be cultured through a seed culture and an intermediate culture.

In one embodiment of the present invention, the seed culture is preferably, but not exclusively, carried out at a temperature of 20 to 40 캜 for 10 to 40 hours after inoculating the lactic acid germ cells into the liquid medium. Depending on the type of the lactic acid bacterium, seed culture may be carried out under non-exhalation conditions.

In one embodiment of the invention, the liquid medium of the seed culture can be adjusted to pH 4.0 to pH 8.0 before sterilization using a previously prepared alkali solution. The composition (% w / v) of the seed culture medium preferably ranges from 2.0 to 10.0 glucose, 0.1 to 5.0 soybean protein hydrolyzate, 0.1 to 5.0 casein hydrolyzate, 0.1 to 5.0 yeast extract, 0.01 to 3.0 dibasic potassium phosphate, 0.1 to 5.0 magnesium sulfate, 0.01 to 1.0 calcium chloride, 0.01 to 0.1 manganese sulfate, 0.01 to 5.0 sodium acetate, but is not limited thereto.

In one embodiment of the invention, the intermediate culture may be carried out between culturing and concentration of the lactic acid bacteria using the seed culture and the first membrane filter as a culture for quantitatively increasing the seed cultured lactic acid bacteria. The intermediate culture may be performed under the same condition as the seed culture, but is not limited thereto. The intermediate culture can be started by inoculating the intermediate culture medium with 0.1 to 5.0%, v / v of the seed culture medium.

In one embodiment of the present invention, the liquid medium of the intermediate culture may be adjusted to pH 4.0 to pH 8.0 before sterilization using a previously prepared alkali solution. The composition (% w / v) of the intermediate culture medium is in the range of 2.0 to 10.0 glucose, 0.1 to 5.0 soybean protein hydrolyzate, 0.1 to 3.0 L-cysteine, 0.1 to 5.0 yeast extract, 0.01 to 3.0 dibasic potassium phosphate, 0.1 To 5.0 manganese sulfate, 0.01 to 0.1 manganese sulfate, 0.01 to 5.0 potassium citrate, 0.01 to 2.0 calcium chloride, 0.01 to 5.0 surfactant, but is not limited thereto.

In one embodiment of the present invention, the lactic acid germ cells cultured through the one or more stages of cultivation or the lactic acid germ cells not having undergone the above-mentioned one or more stages of cultivation are inoculated into the medium, and then the medium inoculated with the lactic acid germs is inoculated into the incubator . Thereafter, the lactic acid germ cells may be cultured and concentrated while passing the culture medium inoculated with the lactic acid germ cells in the first membrane filter.

In one embodiment of the present invention, the culture medium inoculated with the lactic acid bacteria is prepared from a culture medium solution, and the culture medium is prepared by sterilizing impurities or contaminants contained in the culture medium solution through the culture medium filter . Conventional media are usually sterilized by steam or the like. When sterilization of the steam or the like occurs, carbonated impurities such as proteins or the like are generated and remain in the medium, thereby causing a problem of poor quality of the lactic acid bacteria powder. In one embodiment of the present invention, since the feedstock is sterilized through the culture medium filter, impurities due to medium-derived impurities, carbonization or the like generated by conventional steam sterilization do not occur, and thus a high-quality lactic acid bacteria raw material can be provided.

Herein, the culture medium-derived impurities may be heat-denatured non-water-soluble proteins, heat-destroyed vitamins or nutrients, and glucose components decomposed or changed by heat.

The culture medium to which the lactic acid germ cells are inoculated is sufficient if the lactic acid germ cells can be grown and / or cultured. For example, the culture medium contains 1.0 to 10.0% w / v hydrous crystalline glucose, 0.1 to 5.0% w / v soybean protein hydrolyzate , 0.1 to 5.0% w / v yeast extract, 0.01 to 3.0% w / v dibasic potassium phosphate, 0.1 to 5.0% w / v magnesium sulfate, 0.01 to 0.1% w / v manganese sulfate, 0.01 to 2.0% May contain calcium chloride, or may include, but are not limited to, 0.1 to 10.0%, w / v whey powder and 0.1 to 10.0% chicory extract. Further, the medium may further include 0.1 to 5.0%, w / v MgSO ₄ and 0.01 to 0.5%, w / v CaCl ₂ , but is not limited thereto.

In one embodiment of the present invention, the size of the lactic acid germs can be controlled by the shear force applied to the medium while the culture medium inoculated with the lactic acid germs is passed through the first membrane filter. The shear force may be controlled by the flow rate of the medium inoculated with the lactic acid bacteria. The shearing force can be controlled by the channel size (radius) inside the hollow fiber membrane of the first membrane filter through which the culture medium inoculated with the lactic acid bacteria live bacteria, the viscosity of the medium, and the flow rate.

According to the conventional batch-type lactic acid bacteria culture method, it is not easy to control the size of the lactic acid bacteria to be small. In order to solve such a problem, the present invention can easily control the size of the lactic acid bacteria by applying a shearing force to the culture medium containing the lactic acid bacterium to stress the cultured lactic acid bacteria.

It is known that the size of the lactic acid bacteria directly affects the absorption rate of the lactic acid bacteria in the body. Especially, as the size of the lactic acid bacteria is smaller, the absorption efficiency of the lactic acid bacteria increases and the biological efficacy increases.

According to the enrichment method of the present invention, the shearing force applied to the medium can be controlled by controlling the flow rate of the medium passing through the first membrane filter, as shown in Equation 1 above. As a result, the size of the cultured lactic acid germ cells can be easily controlled. Preferably, the size of the live bacteria of the above-mentioned lactic acid bacteria is adjusted to 1.0 탆 or less, thereby maximizing the body absorption rate. As described above, the lactic acid bacterial cells adjusted to have a relatively small size are less aggregated in the subsequent quenching step, and the size thereof is relatively small, so that the absorption in the body can be maximized.

In one embodiment of the present invention, the lactic acid bacteria live bacteria may be concentrated by filtering the impurities contained in the medium inoculated with the lactic acid bacteria live bacteria through the first membrane filter.

In one embodiment of the invention, the size of the lactic acid bacteria can be controlled by passing the lactic acid bacteria live bacteria through a first membrane filter. Further, the impurities in the culture medium can be continuously removed, and the lactic acid bacteria live bacteria can be concentrated.

In the conventional batch-type lactic acid bacteria culturing process, impurities such as proteins, carbohydrates, organic acids, and cell lysates are accumulated in the medium in which the lactic acid germs are inoculated, and the growth of the lactic acid bacteria is restricted by attacking the lactic acid bacteria, Causing a knocking problem.

However, according to the concentration method of the present invention, such impurities can be filtered through the first membrane filter, thereby enhancing the concentration of lactic acid bacteria live cells or dead bacteria. Further, since such impurities are filtered, the washing process can be simplified in the process of collecting the lactic acid bacteria, so that it is possible to reduce costs and simplify the process.

In one embodiment of the invention, the culture medium inoculated with the lactic acid bacteria may be maintained at a pH of 5.5 to 6.8 during the culture of the lactic acid bacteria. The optimal pH of the culture medium inoculated with the lactic acid germs may vary depending on the kind of the lactic acid bacteria. In one embodiment of the invention, when the pH of the medium is lowered by the organic acid produced by the lactic acid bacteria during the culture, a substance that keeps the pH constant can be added to the medium. Examples of the substance that keeps the pH of the culture medium constant include, for example, a sodium hydroxide (NaOH) solution, a potassium hydroxide (KOH) solution, ammonia water, or ammonia gas.

In one embodiment of the present invention, the cultured and concentrated lactic acid bacteria can be killed to produce lactic acid bacteria. The lactic acid bacterium may be strained by heat or a heat treatment, but is not limited thereto.

In one embodiment of the invention, the heat treatment may be carried out at a temperature of 80 ° C to 121 ° C for 3 minutes to 15 minutes. In addition, the heat treatment may be conducted with one to ten times of ultra high temperature sterilization. The ultra-high temperature sterilization may be performed at a temperature of 110 to 130 ° C for 3.0 to 10.0 seconds, for example, twice at 100 ° C for 1.0 second to 10 seconds, at 1.0 to 10.0 at 121 ° C It may be one time per second.

In one embodiment of the present invention, the method for producing dead bacteria of a lactic acid bacteria may further include adding a dispersant to the medium inoculated with the lactic acid bacteria live bacteria after the culture of the lactic acid bacteria live bacteria. The dispersant may exhibit an effect of preventing accumulation phenomenon that may occur during the desizing step of the lactic acid germ viable bacteria.

The dispersant may be added in an amount of 10.0% (w / w) to 80.0% (w / w) based on the pellets of the cultured and concentrated lactic acid bacteria. The dispersing agent may be maltodextrin or trehalose, but is not limited thereto.

In one embodiment of the present invention, the microorganisms of the lactic acid bacteria can be concentrated while passing the culture medium containing the killed microbial bacteria of the lactic acid bacteria as described above through the second membrane filter. When the culture medium containing the killed microbes is passed through the second membrane filter, it is possible to disperse the dead microbes of the lactic acid bacteria, particularly the dead microbes of the microbes accumulated in the process of desizing the lactic acid bacteria, using the shear force applied to the medium have. Further, since the impurities contained in the medium are filtered, it is also possible to wash out the dead bacteria of the above-mentioned lactic acid bacteria.

In one embodiment of the present invention, the culture medium containing the killed microbes may be passed through a second membrane filter to maximize washing and concentration of the killed microbes. By such a concentration method, the concentration of the pathogenic microorganism of the lactic acid bacteria can be preferably about 10 times, 9 times, 8 times, 7 times, 6 times or 5 times that of the microorganism.

Then, the killed bacteria of the lactic acid bacteria may be washed and then dried and pulverized. The powder of dead bacteria of the lactic acid bacteria produced by the concentration method of the present invention can exhibit the effect of preventing the precipitation phenomenon in the solvent. According to the manufacturing method of the present invention, the size of the lactic acid bacteria can be adjusted by the shear force while passing through the first membrane filter. The size-controlled lactic acid bacteria can be aggregated to form lumps during the quenching process, but the masses of dead bacteria of the lactic acid bacteria can be dispersed by the shearing force while passing through the second membrane filter. Therefore, even if the dispersed lactic acid bacteria are pulverized, the aggregation of the dead bacteria of the lactic acid bacteria does not occur, so that the precipitation phenomenon in the solvent can be prevented.

 In one embodiment of the present invention, the culture broth of the lactic acid bacteria may be washed to remove impurities such as proteins, carbohydrates, and lactobacilli except for lactic acid bacteria.

In an embodiment of the present invention, in order to maximize the effect of washing the lactic acid bacteria with the second membrane filter, the washing solution may be added to an incubator, and then passed through the second membrane filter. Here, the washing solution includes, for example, sterilized distilled water and distilled water containing 0.1 to 1.0% w / v sodium chloride (NaCl), but the present invention is not limited thereto. The washing solution may be added at 1.0 to 10 times the volume of the culture medium containing the concentrated microbial cells of the lactic acid bacteria.

In one embodiment of the invention, the drying may be, but not limited to, lyophilization, fluid bed drying or spray drying.

In one embodiment of the present invention, the pulverized lactic acid bacteria dead bacteria may include a size of 0.01 mu m to 3.0 mu m, 0.1 mu m to 3.0 mu m, and 0.5 mu m to 3.0 mu m. Here, 40% to 100% of the powdery lactic acid bacteria dead cells may have a size of 1.0 mu m or less.

In the starter of lactic acid bacteria produced by the concentrating method of the present invention, not only the particle size of the dead bacteria of the lactic acid bacteria is small but also the dead bacteria of the lactic acid bacteria are dispersed evenly. Further, since a relatively small amount of impurities are present through the impurity filtration step by the first membrane filter and the second membrane filter, when the pathogenic bacteria of the lactic acid bacteria are prepared using the concentration method according to the present invention, it is possible to provide a high- have.

When such a high quality Lactobacillus acidophilus strain is added to the food, the functional food or the feed, it does not cause the precipitation phenomenon, so that it can exhibit the excellent flowability and storage effect.

Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited thereto.

[ Example ]

Origin of lactic acid bacteria

L. plantarum (LM1001) and L. plantarum (LM1004) used in this study were isolated from kimchi, a traditional fermented food in Korea, at Korea International University, Department of Pharmaceutical Engineering [Address: 660-759, 965 Dongburo, Munsan-eup, Jinju, Gyeongsangnam-do. The L. plantarum (LM1001) and L.plantarum (LM1004) was deposited with the General Korea Culture Center of Microorganisms (Korean culture center of microorganisms), each new KCCM 42959 (date of deposit: November 12, 2010) and KCCM 43246 (Date of deposit: October 28, 2016).

All lactic acid bacteria used in the following Examples or Experiments are L. plantarum (LM1001) and L. plantarum (LM1004), unless otherwise noted.

2. Analysis method

2-1. Live cell number analysis

The number of viable cells of lactic acid bacteria increased during the culture was analyzed by the colony counting method. At this time, the medium used for the analysis of colonies of L. plantarum was MRS (Difco, USA) solid medium, and cultivation was performed by measuring the number of colonies generated after 48 hours of non-occasional incubation at 37 ° C. The incubator used the JRS-150C model previously used.

The culture was diluted to 10 ⁷ , 10 ⁸ , and 10 ^{9 with a} number of peptones and analyzed using a pouring method using 1.0 ml per petri dish.

The composition (%, w / v) of the peptone water used for the dilution of the medium was as follows: 0.01 to 1.0 sodium chloride, 0.1 to 5.0 casein enzyme hydrolyzate, 0.01 to 5.0 sodium phosphate, 0.01 to 1.0 potassium phosphate (121 ° C, 15 minutes) after sterilization.

2-2. Total bacterial count analysis

After sterilization of the culture medium, the number of dead cells in the culture solution was analyzed using a hemocytometer [Marienfeld, Germany]. The average number N _avg was determined by measuring the number of bacteria in 20 rooms of the hemocyte through a 1,000 magnification optical microscope [BX 53F, Olympus, Japan] ] Were analyzed.

[Formula 2]

Here, R is a dilution factor.

For the analysis of the total number of bacteria present in dead leaves, 1.0 g of dried starch was dissolved in 10 ml of distilled water, and the suspension was well suspended and diluted in sterilized distilled water at 10 ⁸ , 10 ⁹ and 10 ¹⁰ .

2-3. HPLC Analysis of Glucose and Lactic Acid

HPLC (Agilent Technology 1200, Agilent, USA) was used for the analysis of organic acids such as glucose and lactic acid during the culture. (NH _2, 300 mm × 7.8 mm, 9.0 mm particle size, Bio-Rad, USA) and 5 mM H ₂ SO ₄ solution (0.5 ml flow rate) for the stationary phase and the mobile phase, Were used. The temperature of the column was maintained at 35 ° C during the analysis and the sample was automatically injected through an autosampler (5.0 mL). Analysis was performed using an Agilent Chemstation 1200 analysis program using an RI detector.

2-4. Immuno-active assay method

Immunoreactivity and cytotoxicity of heat-killed probiotics (HK-probiotics) were measured by in-vitro analysis using immunocytes (RAW 264.7) and mouse spleen (splenocytes) 12), interleukin -4 (IL-4), interferon-gamma (INF-γ), TNF- alpha (TNF-α), NO ^- (Nitric oxide), viability (viability) and proliferative changes in the immune cells and the production .

2-4-1. Sample collection

Lactic acid bacteria and lactic acid bacteria samples prepared by the method of the present invention were prepared using L. plantarum (LM1001).

Lactic acid bacteria were analyzed by an in-vivo test in which the diluted live cells were orally administered at a fixed concentration to measure changes in cytokines in the mouse blood. Immunoglobulin activity of the killed cells was determined by measuring the splenocytes and macrophages (RAW264.7 ) Were used to analyze the changes of cytokines after in vitro treatment of dead cells.

2-4-2. Experimental animals and treatment

Six-week-old male Balb / c mice were purchased from Samtaco Korea (Korea). The feeding environment was maintained at a temperature of 22 ± 2 ° C and a humidity of 50 ± 20% for 12 hours. Feeds were fed with solid feed for mouse and water for drinking water without restriction. The animals were adapted for one week in an experimental animal room and then subjected to experiments.

Lactic acid bacteria of L. plantarum (LM1001) prepared by the method of the present invention was orally administered once a day for 10 days for each concentration. Blood samples were collected from the experimental animals 24 hours after the last oral administration and stored at -80 ° C. Con A was intravenously administered at 25 mg / kg as a positive control.

2-4-3. Isolation and culture of mouse splenocytes

The spleen was aseptically removed from the mouse, washed with RPMI 1640 solution, and then pulverized to obtain cells. The separated cell suspension was passed through 200 mesh stainless steel, centrifuged at 1,200 rpm for 3 minutes at 4 ° C, and the cell pellet was suspended in ACK buffer for 5 minutes to remove red blood cells. The spleen cells were suspended in RPMI 1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin at a concentration of 1 × 10 ⁶ cells / ml, and 500 μl of each was dispensed into a 48-well plate. Lactobacillus acidophilus L. plantarum produced by the present method was treated by concentration. Lipopolysaccharide and Con A were treated with cells as a positive control.

Cells treated as above were cultured in a 5% CO ₂ incubator at 37 ° C for 3 to 72 hours. The supernatant of the culture was stored at -20 ° C and used for cytokine production.

2-4-4. Cell culture

Spleen cells and RAW 264.7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 100 units / mL of streptomycin and penicillin at 37 ° C and 5% CO ₂ to determine the effect on NO and cell proliferation Respectively.

2-4-5. Cytokine ( IFN -γ, TNF -α, IL-12) Secretory function  Measure

After splenocytes were cultured, secretion of cytokines (TNF-α, IFN-γ, IL-12) secreted from the culture supernatant was measured. After L. plantarum (LM1001) prepared by the method of the present invention was treated with positive control LPS and Con A for a certain period of time, the concentration of free cytokine in the culture solution was measured by enzyme-linked immunosorbent assay ELISA) method (ELISA kit, R & D system, USA).

That is, 100 μl of the supernatant sample was added to a 96-well plate coated with an antibody of cytokine, allowed to react at room temperature for 2 hours, and then the supernatant solution was removed. The supernatant was washed three times with a phosphate buffer solution and Tween-20 (Sigma) Lt; / RTI >

Then, a solution containing the secondary antibody was added to react with the primary antibody, and Horseradish peroxidase (HRP) enzyme conjugated with avidin was added thereto, followed by reaction at room temperature for 15 minutes. After that, TMB solution was added as a substrate for the HRP enzyme, and the reaction was confirmed by color change.

The change in color was observed depending on the concentration of cytokine present in the sample. Thus, the presence or absence of cytokine production in the sample was confirmed by this change. The reaction between the HRP enzyme and the TMB substrate was terminated by adding sulfuric acid (1.0 M), and the absorbance was measured at 450 nm using a microplate reader (Thermo).

2-4-6. NO measurement

The macrophage RAW 264.7 cells were seeded in a 96 well plate at a concentration of 5 × 10 ⁵ cells / ml, cultured for 24 hours, treated with 1 mg / ml of LPS as a positive control, and treated with phosphate buffer solution as a negative control. A sample of L. plantarum (LM1001) prepared by the method of the present invention was treated and then re-cultured for 24 hours.

The NO concentration was measured using a Griess reagent system (Sigma, USA). 50 μL of the culture was added to 96-well plate, and the same amount of Glycine Agent I (NED solution) and Glycyrrhizin II (sulfanilamide solution) were mixed in a dark room. After 10 min of incubation in a dark room, microplate reader (Tecan, Austria) at 540 nm. The NO concentration was calculated using a standard curve of sodium nitrite (0 to 100 micromolar).

2-4-7. Measurement of cell proliferation rate

The proliferation rate of splenocytes and RAW 264.7 cells was determined by WST-1 assay. The splenocytes and RAW 264.7 cells were adjusted to a concentration of 5 × 10 ⁵ cells / well and dispensed in a 96-well plate, followed by treatment with a sample and a control group and culturing for 48 hours. 100 ㎕ of WST-1 kit solution was added and incubated for 1 hour. Absorbance was measured at 540 nm using an ELISA reader (Thermo, Germany).

[Formula 3]

Here, N (%) is the cell proliferation ratio, C _p is the absorbance after the fungus treatment, and C _s is the absorbance after the untreated treatment.

2-4-8. Statistical processing

The results were analyzed by one way ANOVA test using the SPSS 21 manual (Statistical Package for Social Sciences, IBM, Armonk, NY, USA) T test at the P < 0.05 level.

2-5. Analysis of lactobacillus anti-obesity effect

In order to confirm the effect of inhibiting the accumulation of fat in the adipocyte, a sample of Lactobacillus koreanum prepared according to the embodiment was induced to differentiate into a mature fat cell in the lipocortical cell (3T3-L1 cell) O) staining and intracellular triglyceride content (TG kit).

2-5-1. Inhibition effect of fat precursor cell growth

MTT assay (Methylthiazolinyldiphenyl tetrazolium bromide assay) was performed to analyze the cytotoxicity of lactic acid bacteria in the culture medium of 3T3-L1 (mouse embryonic fibroblast-adipose like cell line) to analyze the anti-obesity effect of lactic acid bacteria.

3T3-L1 adipocytes were plated at 4 × 10 ⁴ cells / ml in 96-well plates (96-well plate) and cultured in a 5% CO ₂ , 37 ° C. incubator for 24 hours. After the culture, the culture medium was removed and DMEM medium and lactic acid bacteria powder were treated for 24 hours at concentrations of 0, 100, 250 and 500 ㎍ / ml. Then, a solution of MTT (3- [4,5-dimethyl-thiazole] -2,5-diphenyl-tetrazolium bromide) dissolved in 5 mg / ml of DBS in a solvent DPBS (Dulbecco's Phosphate-Buffered Saline, And incubated for 4 hours in a 5% CO ₂ incubator at 37 ° C.

After the culture, the culture solution was removed, and dimethyl sulfoxide (DMSO) was added to each well at a rate of 200 μl / well and mixed well using a pipette. The absorbance was then measured at 570 nm with an ELISA reader.

2-5-2. Measurement of adipocyte lipid accumulation inhibitory activity (Oil Red O staining analysis)

3T3-L1 adipocytes were fed to a 6-well plate (6-well plate) at a density of 7 × 10 ⁴ cells / ml and differentiated into adipocytes for 8 days, / ml). After 8 days, the cell culture was discarded and washed with phosphate buffered saline (PBS). Then, 500 μl of 10% formaldehyde was added to each well, fixed at 4 ° C for 1 hour, washed with phosphate-buffered saline three times, and dried in a CO ₂ incubator after removal of formaldehyde.

After the plates were dried, 500 μl of Oil Red Dye was added, and the mixture was stained for 30 minutes at room temperature in a dark state, and washed three times with phosphate-buffered saline. The stained cells were observed under a microscope. After observation, the stained dye in the adipocyte was extracted with 300 μl of iso-propanol per well and the optical density value (OD value) was measured at 500 nm with an ELISA reader.

The oil red dyes were obtained by mixing 500 mg of Oil Red dyestuff with 100 ml of iso-propanol in distilled water at a ratio of 6: 4 and filtering with 0.45 ㎛ filter.

2-5-3. Analysis of triglyceride contents in adipocytes

To 7 × 10 ⁴ cells / ml density and a frequency divider, while differentiation of fat cells for 8 days at the same time, the lactic acid bacteria powder, dead cells at different concentrations (100, 250, 500 3T3-L1 (6-well plate), the fat cells 6-well plates Mu] g / ml).

After 8 days, each well was washed with phosphate-buffered saline, cells were collected using a cell scraper (SPL), and the intracellular triglyceride was extracted using an ultrasonic pulverizer (SONIFIER 450, BRANSON) -S, ASAN PHARMACEUTICAL Co., Ltd.) was used to measure the content of triglycerides.

Example 1 Culture and Concentration of Lactic Acid Bacteria

1-1. Storage of lactic acid bacteria

The lactic acid bacteria used in the examples of the present application were first cultured at 37 ° C. for 48 hours under anaerobic conditions using MRS (Difco, USA) solid medium. After 48 hours, the lactic acid bacteria collected on the medium were harvested in sterile vats and washed 3 times with phosphate buffered saline (PBS, pH 6.8), followed by addition of appropriate cryoprotectant (25% glycerin + 10% (0.2 ml) was dispensed into a cryo-vial (1.2 ml, Simport, Canada) prior to freezing in a -70 ° C cryogenic freezer.

The defreezed vials (DF vials) were stored for a maximum of 6 months, taken out and melted as needed. All experiments were conducted in aseptic conditions to minimize contamination.

1-2. Lactobacillus Seed culture

The vial was taken out from a cryogenic freezer and dissolved, followed by seed culture of lactic acid germ.

First, DF-vials (0.2 ml) were inoculated into sterile 15 ml MRS (Difco, USA) liquid medium (18f, laboratory) and incubated at 37 ° C for 12 hours in an incubator [JSBI-150C, JSR Korea] .

Secondly, 15 ml of the lactic acid bacteria cultured for 12 hours was inoculated again with 1.0 L seed culture medium LTMS-PR-SM (2.0 L Erlenmeyer flask, 4) and incubated at 37 ° C for 10 hours in an aseptic condition [JSRB-150C, JSR Korea].

The composition (%, w / v) of the seed medium LTMS-PR-SM used at this time is as follows: 2.0-10.0 hydrolyzed glucose, 0.1-5.0 soybean protein hydrolyzate, 0.1-5.0 casein hydrolyzate, 0.1-5.0 yeast extract , 0.01 to 3.0 divalent potassium phosphate, 0.1 to 5.0 magnesium sulfate, 0.01 to 1.0% calcium chloride, 0.01 to 0.1 manganese sulfate, 0.01 to 5.0 sodium acetate and the like. Respectively.

1-3. Medium culture of lactic acid bacteria live cells

The 1.5 L seed culture cultivated in the seed culture was used to inoculate the 80 L intermediate culture medium LTMS-PR-MM (100 L fermenter, Kobioctak). This intermediate culture was carried out for 12 hours with slight agitation at 32 DEG C and ammonia gas was added periodically whenever necessary to adjust the pH of the culture medium from pH 5.0 to pH 7.0.

The composition (%, w / v) of the intermediate culture medium used in this case is as follows: 2.0-10.0 hydrolyzed glucose, 0.1-5.0 soybean protein hydrolyzate, 0.1-3.0 L-cysteine, 0.1-5.0 yeast extract, 0.01 , Sodium hydroxide (10.0M) prior to sterilization, sodium hydroxide (0.01M), sodium hydroxide (0.01M), potassium hydroxide (0.01M NaCl) and adjusted to pH 6.8.

1-4. Cultivation and concentration of lactic acid bacteria using first membrane filter

The bioreactor used in the concentrating method of the present invention includes a first membrane filter (hollow-sieve filter having a pore size of 0.5 mu m diameter and a 6 mm channel radius, Al-Al ceramic hollow fiber membrane filter) and an incubator, The culture solution was circulated through the membrane filter and the incubator by a forced aseptic circulation pump (Q = 9.0 m ³ / h, H _max = 41 m, 5.0 bar, 120 ° C) .

First, the culture medium was inoculated with 20 L of the culture medium inoculated with the cultured lactic acid bacterium via the seed culture and the intermediate culture. Thereafter, the culture medium containing the lactic acid germ cells was passed through the first membrane filter using a forced aseptic circulation pump, followed by concentration and filtration.

Through the above-described concentration and filtration, the intermediate culture medium was filtered, and the new medium was added to the incubator. The composition (%, w / v) of the freshly inoculated medium is as follows: 2.0-10.0 hydrolyzed glucose, 0.1-5.0 soybean protein hydrolyzate, 0.1-5.0 yeast extract, 0.01-3.0 potassium phosphate, 5.0 Magnesium sulfate, 0.01 to 0.1 manganese sulfate, 0.01 to 2.0 calcium chloride. The freshly inoculated medium was pre-sterilized through a media filter (Al-Al ceramic hollow fiber filter with a pore size of 0.2 탆 diameter and a 6 mm channel radius) before being added to the incubator.

After the whole medium was replaced with freshly added medium, the incubator was cooled to 18 캜 or lower, and the flow of the culture fluid flowing along the first membrane filter was kept to a minimum. The pH of the culture medium was maintained at 5.0 to 6.8 (1: 1 = 20%, w / v ammonia water: distilled water) for 5 hours until the culture and concentration of the lactic acid bacteria viable.

Thereafter, the temperature of the bioreactor is raised to 32 캜 before starting the cultivation and concentration of the lactic acid germ viable bacteria, and the culture solution containing the medium inoculated with the lactic acid germ viable bacteria is passed through the first membrane filter, &Lt; / RTI &gt; At this time, the volume of the culture liquid decreases at a constant rate as it passes through the first membrane filter. The freshly inoculated medium is sterilized by a pump (YZ15, Leadfluid, China, Max = 20.0 L / h) Followed by feeding.

The flow rate of the culture medium circulating along the first membrane filter was adjusted to 50.0 L / h to 300.0 L / h using the forced aseptic circulation pump. Ammonia water (1: 1 = 20% w / v ammonia water: distilled water). The internal pressure of the fermenter was maintained at 0.1 to 0.5 kg / cm ² using nitrogen gas to maintain the aseptic state.

Example 2: Strains of lactobacillus &lt; RTI ID = 0.0 &gt;

In one embodiment of the present invention, prior to saccharification of the lactic acid germ viable cells obtained in Example 1, the medium inoculated with the lactic acid germs inoculated is divided into Group 1 in which no dispersing agent is added, 0.1 to 80.0% by weight of maltodextrin as a dispersing agent, And Group 2 and Group 3 to which 80.0% by weight of trehalose was added, respectively.

In one embodiment of the present invention, lactic acid bacteria obtained in Example 1 were circulated for 3 seconds at 121 DEG C for 3 seconds using an ultrahigh-temperature continuous sterilizer to prepare lactic acid bacteria dead bacteria (Example 2-1).

In one embodiment of the present invention, lactic acid bacteria obtained in Example 1 were circulated at 110 DEG C for 5 minutes using an ultra-high temperature continuous sterilizer to produce lactic acid bacteria (Example 2-2, FIG. 5B).

In one embodiment of the present invention, lactic acid bacteria obtained in Example 1 were circulated for 3 minutes at 65 DEG C, 85 DEG C, and 120 DEG C using an ultra-high temperature continuous sterilizer to produce lactic acid bacteria dead cells (Examples 2-3, 7).

In one embodiment of the present invention, the lactic acid germs obtained in Example 1 were circulated at 80 ° C for 0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes, respectively, to produce lactic acid bacteria. , Fig. 9).

Example  3. Lactic acid bacteria Dead bacteria Second membrane  Concentration, washing and dispersion using filters

The culture medium containing the lactic acid bacteria obtained in Example 2-1 was filtered through a second membrane filter (having a pore size of 0.01 mu m to 0.1 mu m in diameter and a channel radius of 3 mm and having a molecular weight of 50,000 daltons to 500,000 daltons And passed through a polysulfone hollow fiber membrane filter capable of discharging it, to concentrate the dead bacteria of the lactic acid bacteria 2 to 10 times. While using the second membrane filter, a shearing force was applied to the cultured liquid of the lactic acid bacteria, which was collected in the desizing step of Example 2, to disperse the dead bacteria of the lactic acid bacteria.

In addition, in order to maximize the effect of the lactic acid bacteria dispersion by the second membrane filter, a dispersant was added to the culture medium, and all results are shown in Table 2, FIG. 8, and FIG.

As shown in Fig. 8, when the bacteria of the lactic acid bacteria are concentrated and washed using the second membrane filter, It was found that the accumulated strains of lactic acid bacteria were dispersed and concentrated and recovered in the heat treatment step. Further, even when the second membrane filter is used, it is a matter of course to those skilled in the art that the impurities are filtered and removed from the culture liquid as in the first membrane filter.

As described above, lactic acid bacteria are clustered in the process of saccharification to form aggregates of lactic acid bacteria dead cells. In the present example, the size of the mass of dead bacteria of the lactic acid bacteria formed immediately after the quenching of the lactic acid bacteria was 20 占 퐉 To 200 mu m (Fig. 9). The mass of dead bacteria of the lactic acid bacteria or the dead bacteria of the lactic acid bacteria immediately after the quenching was passed through the second membrane filter to disperse the agglomerated mass of the dead bacteria of the lactic acid bacteria. The size of the dispersed lactic acid bacteria dead cells was measured to be 0.5 탆 to 3.0 탆 or less (Fig. 9).

Example  4. Lactic acid bacteria Dead bacteria  Washing, drying and Powdered

After the concentration of the dead bacteria of the lactic acid bacteria according to the above Example 3, distilled water sterilized as a washing solution was added to the incubator, and the lactic acid bacterial strain was washed by passing it through the second membrane filter.

The sterilized distilled water was added twice as much as the culture medium containing the concentrated Mycobacterium tuberculosis bacteria, and then the diluted culture medium was again passed through the second membrane filter to concentrate the diluted medium to a volume before dilution. As a result, it showed the effect of washing germs concentrated with sterilized distilled water of a total volume of 6 times the volume of the medium containing the concentrated dead cells.

The killed bacteria of the lactic acid bacteria washed by the above method were dried in a freeze dryer for a total of 96 hours to be powdered.

The size distribution of washed, dried and powdered lactic acid bacteria dead cells was measured and shown in FIG. As shown in FIG. 9, it was found that the size of the powdered dead cells showed a size distribution of 0.5 μm to 3.0 μm, and further, the size of 60% to 100% of the powdered dead cells appeared to be 1.0 μm or less . This is because the lactic acid bacteria which had increased to 40 ~ 200 쨉 m in the heat treatment process were dispersed by the shearing force applied to the culture liquid by the second membrane filter.

Comparative Example 1. Preparation of lactic acid bacteria dead cells using batch culture

Batch cultivation of lactic acid bacteria was carried out in a total volume of 80 L in a 100 L fermenter (Kobioctak). First, 4.0 L (5.0%, v / v) of the culture liquid containing the lactic acid germ cells cultured through the seed culture was inoculated into the fermentation tank into the 100 L fermenter.

During the culture in the batch incubator, the stirring speed was 30 rpm and the temperature was maintained at 32 캜. In addition, nitrogen gas was used to maintain the ash state. During the culture, the pH was maintained at pH 5.0 to pH 7.0 using ammonia gas. During the batch culture, the live bacteria of the lactic acid bacteria were sampled and analyzed at regular intervals.

Thereafter, in one embodiment of the present invention, the live bacteria of the above-mentioned lactic acid bacteria were heat-treated at 110 DEG C for 5 minutes and sacrificed (Comparative Example 1-1, Fig. 5). As shown in FIG. 5, the dead bacteria of the lactic acid bacteria produced using the conventional batch culture was larger and less concentrated than the dead bacteria of the present invention.

Thereafter, in one embodiment of the present invention, the live bacteria of the above-mentioned lactic acid bacteria were circulated for three times at 120 DEG C for 3 seconds to be sacrificed (Comparative Example 1-2, FIG. 7). As shown in Fig. 7, the dead bacteria of the lactic acid bacteria produced using the conventional batch culture method was observed to be larger than the bacteria of the lactic acid bacteria according to the present invention.

In addition, the dead bacteria of the lactic acid bacteria was dried and pulverized in the same manner as in Example 4.

[Experimental Example]

Experimental Example 1. Evaluation of Productivity and Growth Rate of Lactic Acid Bacteria

The productivity and growth rate of the lactic acid germ cells according to Example 1 and Comparative Example 1 are shown in Table 1 below. The productivity of the lactic acid bacterium was expressed by the amount of pellet, and the specific growth rate (m) of the lactic acid bacterium was expressed by the following equation (4).

[Formula 4]

Here, LN is the logarithmic function formula, t ₁ is the concentration of the lactic acid bacteria at the specific time t ₁ , t ₂ is the concentration of the lactic acid bacteria at the specific time t ₂ , and t is the elapsed time.

[Table 1]

Here, feeding rate (h ^-1 ) means a rate at which the freshly inoculated medium is fed to the incubator before culturing and concentration of the lactic acid bacteria live bacteria by the first membrane filter after the intermediate culture in Example 1 , And analyzed according to Equation 5 below.

[Formula 5]

Where Q _h is the amount of medium consumed in one hour (L), and V is the total volume (L) of the bioreactor.

As shown in Table 1, the amount of pellets of the lactic acid bacterium according to Comparative Example 1 was 18.3 g / L (LM1001) and 15.2 g / L (LM1004), respectively, (LM1001) and 170.0 g / L (LM1004), respectively.

The culture broth of the lactic acid bacterium according to Comparative Example 1 had a lactic acid concentration of 478.1 mM (LM1001) and 534.1 mM (LM1004), respectively, whereas the culture solution of the lactic acid bacterium according to Example 1 had a lactic acid concentration of 324.3 mM ) And 369.2 mM (LM1004).

As a result, it was found that impurities such as lactic acid were continuously filtered and removed through the first membrane filter and kept constant during the culture in the manufacturing method according to Example 1. Thus, it was found that the impurities were maintained at a constant concentration, thereby increasing the amount of the final production of the lactic acid bacteria as compared with Comparative Example 1.

Experimental Example 2. Evaluation of Size Control of Lactic Acid Gonococci

FIG. 4 shows a photograph of the lactic acid germ cells present in the powder after the recovery of the lactic acid germs according to Example 1 and Comparative Example 1 by drying and pulverization and scanning with a scanning electron microscope (SEM, X10,000) , And a photograph taken with an optical microscope (X1, 000) are shown in Fig.

The size distribution of the lactic acid germ cells according to Example 1 and Comparative Example 1 was measured and shown in FIG.

As shown in FIG. 4 and FIG. 5, it was found that the lactic acid germ viable bacteria according to Example 1 were not only small in size but also more concentrated than those in Comparative Example 1.

Further, as shown in Fig. 6, the size of the lactic acid germs according to Comparative Example 1 was 6.3 mu m maximum, while the size of the lactic acid germs according to Example 1 was smaller than 4.0 mu m Respectively. In addition, when the lactic acid germ cells according to Example 1 were heat-treated and homogenized, the size decreased to 3.0 μm, which is significantly smaller than 4.0 μm in Comparative Example 1.

Experimental Example 3. Evaluation of Size Control of Lactic Acid Bacteria

The characteristics using the second membrane filter according to Example 3 and the characteristics using centrifugation as a comparative example are shown in Table 2 below in the process of recovering bacteria from the lactic acid bacteria according to Example 2-1.

[Table 2]

As shown in Table 2, it was found that the cumulative yield was increased to 115.2% (Group 1), 148.5% (Group 2), and 168.7% (Group 3) in Example 3. However, when the dead bacteria of the lactic acid bacteria were recovered by centrifugation according to the comparative example, the accumulation of bacteria became more severe, and the cumulative yield was rapidly decreased to 26.4%.

Experimental Example  4. Lactic acid bacteria Dead bacteria Suspension  evaluation

The present experiment was carried out to observe the stability of the host suspension of lactic acid bacteria according to Example 4 and Comparative Example 1-1.

Experiments were carried out on Lactobacillus plantarum (LM1001) lactic acid bacteria using 1.0E + 12 / g native horse according to Example 4 and Comparative Example 1-1. Thereafter, the dead organism-derived dead organism seeds of Example 4 were suspended in distilled water sterilized at a concentration of 1.0E + 8 / ml and the dead organism-derived dead organism seeds of Comparative Example 1-1 at a concentration of 1.0E + 7 / ml, And the precipitated state was observed (Fig. 11).

The density of germ cells of the lactic acid bacteria prepared by the above enrichment method was 5.2E + 12 / g, and the density of germs of the lactic acid bacteria prepared through the batch culture was 2.9E + 12 / g was the original.

As shown in Fig. 11, in the case of batch culture, a part of the suspension was precipitated within one hour of suspension, whereas in the case of the manufacturing method of the present invention, the suspension was stably maintained for 4 days after suspension And the like.

Experimental Example  5. Immunological activity analysis of lactic acid bacteria

Immunoreactivity of two strains of L. plantarum prepared by the enrichment method of the present invention was analyzed for each of live cells and dead cells.

The immunoactivity of the lactic acid bacteria was assayed as follows: After feeding the feed containing the lactic acid bacteria live cells, changes in cytokine (TNF-α, INF-γ, IL-12) The in-vivo test was carried out and is shown in Fig.

Specifically, two 6-week-old male Balb / c mice were orally administered with two kinds of lactobacillus live bacteria per concentration (10, 100 mg / kg) every day for 10 days. Blood samples were collected from the experimental animals 24 hours after the last oral administration and stored at -80 ° C before use in the experiment. Con A was intravenously administered at 25 mg / kg as a positive control.

As shown in Fig. 12, the amount of serum cytokine after the lactobacillus treatment for 10 days was measured by ELISA assay. TNF-α, IFN-γ, and IL-12 were significantly increased compared to the positive Con A (25 mg / kg).

Experimental Example 6. Analysis of Immunological Activity of Mycobacterium tuberculosis

The immune activities of two species of L. plantarum prepared by the enrichment method of the present invention were analyzed.

The immunological activity of the lactic acid bacteria was analyzed as follows: RAW264.7 cytotoxicity experiment using macrophages, production of NO ^- (nitric oxide), and cytokine (TNF-α, INF -γ, IL-12, and IL-6) was performed and shown in FIG.

The cytotoxicity and inducibility of Nitric oxide production of lactic acid bacteria using RAW264.7 macrophages were investigated according to the procedure described in the previous method.

Fig. 13 (A) shows the results of an experiment of cytotoxicity after treating a lactic acid bacterium sample for 48 hours in order to set the cell treatment concentration of lactic acid bacteria in RAW 264.7 cells, which is a mouse macrophage cell line. Indicating no cytotoxicity. Therefore, the immunoactivity of lactic acid bacteria was measured at a concentration (0.1-10 ㎍ / ㎖ or less) at which no cytotoxicity was observed. Cytotoxicity was measured by the activity of the liberated lactate dehydrogenase.

Fig. 13 (B) is a graph showing the effect of Lactobacillusthus koreanum powder treatment on the NO production ability of mouse macrophage cell line Raw 264.7 cells. To the RAW 264.7 cells, lactobacillus samples were treated at 0.1, 1 and 10 ug / ml for 48 hours , And the activity of NO released in the culture medium was measured using a Griess reagent system.

As shown in Fig. 13, the ability of NO to be increased by the positive control group LPS was increased about 6 times, and it was confirmed that the concentration of NO was significantly increased in all of the lactic acid bacteria treated.

Fig. 14 shows the effect of microbial cells of lactic acid bacteria in the production of cytokines using mouse spleen cells. Fig. 14 (A) shows that splenocyte cytotoxicity does not appear at a concentration of 0.1 to 10 mg / ml of the above-mentioned lactic acid bacteria dead powder. 14 (B), 14 (C), 14 (D) and 14 (E) show that treatment of the lactic acid bacteria dead bacteria powder increases the production of cytokines with a significant concentration.

As shown in Figs. 12 to 14, both of the lactic acid bacteria of the two species were found to be immunopotentiating in both live bacteria and dead bacteria, indicating that they exhibited immunological activity.

Experimental Example 7. Anti-obesity effect of strains of lactic acid bacteria

In order to observe the anti - obesity effect of Lactobacillus koreanum, fat accumulation inhibition experiment was performed. Specifically, 25 mg / ml con A, a fat accumulation inducer, was added to the adipocytes, and the powder of L. planatrum (LM1004) lactic acid bacteria was treated at different concentrations (0, 100, 250, 500 ㎍ / ml) The amount of fat accumulation was measured. As a control group, the group in which the fat accumulation inducer was not treated and the group in which the fat accumulation inducing substance was treated and then the lactobacillus microorganism powder was not treated were used. As shown in FIG. 15, , 100, 250, 500 占 퐂 / ml), it was observed microscopically that the amount of lipid accumulated in adipocytes was decreased in a concentration-dependent manner (Fig. 15A).

Especially, at the concentration of 500 / / ml, it was confirmed that the lipid in the adipocyte was reduced by about 46% when compared with the positive control (Con A) (Fig. 15C).

In addition, the amount of triglyceride accumulated in the adipocytes was decreased according to the treatment concentration (0, 250, 500 ㎍ / ml), especially when compared with the positive control (Con A) at 500 ㎍ / It was confirmed that the amount of triglyceride (TG) in fat cells was reduced by about 81% (FIG. 15D).

Experimental Example 7 showed that LM1004 also exhibited lipid accumulation inhibition in adipocytes.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Korea Microorganism Conservation Center KCCM43246 20161028 Korea Microorganism Conservation Center KCCM42959 20101112

Claims (11)

As a method for concentrating microorganisms of lactic acid bacteria,
Preparing lactic acid bacteria live bacteria;
Homogenizing the lactic acid germ cells by tin or heat treatment;
Preparing a lactic acid bacterium culture liquid comprising a medium containing a mass of killed microbial bacteria formed by clustering of the microbial strains of the lactic acid bacteria in the step of homogenizing the lactic acid bacteria; And
The above-mentioned lactic acid bacteria culture solution is passed through a membrane filter including a hollow fiber membrane filter containing pores having a diameter of 0.01 mu m to 0.1 mu m to disperse the above-
Impurities other than lactic acid bacteria contained in the culture medium are filtered through the membrane filter
And a method of concentrating the pathogenic bacteria of lactic acid bacteria.
delete The method according to claim 1,
Wherein the microorganism of the present invention is a microorganism belonging to the genus Escherichia.
A method for concentrating lactic acid bacteria dead bacteria.
delete delete The method according to claim 1,
Wherein the membrane filter comprises a hollow fiber membrane filter for filtering a material having a molecular weight of 50,000 daltons to 500,000 daltons.
A method for concentrating lactic acid bacteria dead bacteria.
The method according to claim 1,
Wherein the lactic acid bacterium comprises a bacterium, a bacterium, a non-bacteriophage, and a combination thereof.
A method for concentrating lactic acid bacteria dead bacteria.
The method according to claim 1,
Wherein the lactic acid bacterium comprises Lactobacillus plantarum .
A method for concentrating lactic acid bacteria dead bacteria.
9. A method for concentrating a microorganism of the genus Escherichia coli according to any one of claims 1, 3 and 6 to 8,
A method for producing lactic acid bacteria dead cells.
The lactic acid bacteria killed by the method according to claim 9.
An additive comprising the Mycobacterium tuber of claim 10,
Wherein the additive can be added to food, functional food or feed,
additive.

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