WO2018101612A1 - Method for controlling size of lactobacillus using membrane filter - Google Patents

Abstract

The present application relates to a method for controlling the size of lactobacillus comprising passing a lactobacillus culture solution through a membrane filter.

Description

Lactic Acid Bacteria Size Control Using Membrane Filter

The present application relates to a method for controlling the size of lactic acid bacteria comprising passing the lactic acid bacteria culture medium through a membrane filter.

The definition of probiotics specified by the World Health Organization (WHO) is a live microorganisms that, when administered in adequate amounts, confer a health benefit on the host). However, as the commercialization of probiotics of lactic acid bacteria has rapidly increased and increased, microorganisms are also used as probiotics in the probiotic market.

Probiotics as microbial cells have various advantages over probiotics and are already commercially available in Japan and the United States. Probiotics can be used more stably than probiotics, so they have a wide range of industrial applications, are easy to handle in the distribution process, and are added to general foods to enhance the functionality of foods. In recent years, the market has increased significantly.

As a conventional technique of the method for producing bacterium, there is a batch culture method. The batch culture method, after the culture of lactic acid bacteria growth lactic acid bacteria and lactic acid bacteria growth in a large-capacity medium, comprising the step of heating and culturing the cultured lactic acid bacteria (Korean Patent No. 10-2012-0047792) .

However, when the dead bacteria are prepared by conventional batch culture, the size of the lactic acid bacteria cannot be adjusted. The smaller the size of the lactic acid bacteria is known to increase the absorption rate in the body, it is known to increase the biological efficacy. Accordingly, in order to increase the absorption rate of lactic acid bacteria in the body, the development of a method for producing lactic acid bacteria while controlling the size of the lactic acid bacteria in the manufacturing process of lactic acid bacteria has been continuously studied.

In order to solve the disadvantages of the prior art as described above, the present application is to provide a method for controlling the size of lactic acid bacteria, including passing the lactic acid bacteria culture medium through a membrane filter.

However, the problem to be solved by the present application is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The first aspect of the present application provides a method for controlling the size of lactic acid bacteria comprising passing the lactic acid bacteria culture medium through a membrane filter.

2nd aspect of this application provides the manufacturing method of lactic acid bacteria containing said size adjustment method.

The third aspect of the present application provides a lactic acid bacterium produced by the production method of the lactic acid bacteria.

The fourth aspect of the present application provides an additive containing the lactic acid bacteria.

According to the embodiments of the present invention, by applying a shear force to the lactic acid bacteria using the membrane filter, it is possible to easily control the size of the lactic acid bacteria, in particular it is possible to adjust the size of the lactic acid bacteria small.

Accordingly, lactic acid bacteria prepared using the control method of the present application may not only be effectively absorbed into the body but also effectively absorbed into the body, thereby exhibiting excellent immune activity and anti-obesity effect.

1 is a schematic diagram of a method for producing lactic acid bacteria including lactic acid bacteria control method according to an embodiment of the present application.

Figure 2 is a schematic diagram comparing the structure of the bioreactor (a) including the incubator and membrane filter according to an embodiment of the present application and the conventional batch lactic acid bacteria incubator (b).

Figure 3 is a graph showing the productivity, growth rate, and concentration of lactic acid bacteria live bacteria cultured and concentrated according to an embodiment of the present application (LM1001, LM1004) and lactic acid bacteria live bacteria cultured according to Comparative Example 1.

FIG. 4 is an electron micrograph of a lyophilized lactic acid bacterium bacterium produced according to Comparative Example 1 and a lactic acid bacterium bacterium prepared according to Comparative Example 1 in a scanning type comparison (SEM, X10,000).

Figure 5a is a photograph comparing the size of the lactic acid bacteria live bacteria cultured and concentrated according to an embodiment of the present application (right) and the lactic acid bacteria live bacteria (left) cultured according to Comparative Example 1 (optical, X1,000).

Figure 5b is a photograph comparing the size of the lactic acid bacteria bacteriophage (right) and the lactic acid bacteria bacterium (left) prepared according to Comparative Example 1 in accordance with an embodiment of the present application (optical, X1,000).

6 is a graph comparing the size distribution of lactic acid bacteria living bacteria cultured and concentrated according to an example of the present application with lactic acid bacteria living bacteria cultured according to Comparative Example 1. FIG.

Figure 7 is a graph comparing the size distribution of the lactic acid bacteria killing lactic acid bacteria and the lactic acid bacteria killing bacteria prepared according to Comparative Example 1 according to an embodiment of the present application.

8, in the production method according to an embodiment of the present application, immediately after incubation and concentration of lactic acid bacteria (left: LM1001, right: LM1004) (first line), immediately after sterilization (second line), immediately after concentration and washing (third) Joules) are photos showing the size of each lactic acid bacteria (optical, X1,000).

9 is a graph comparing the size change before and after passing the lactic acid bacteria killing bacteria produced according to an embodiment of the present application to the second membrane filter.

Figure 10 is a photograph comparing the properties of the original, that is, chromaticity of the lactic acid bacteria bacterium produced according to one embodiment (left) and Comparative Example (right) of the present application.

Figure 11 shows a comparison of the results of suspending the original powder of lactic acid bacteria killed according to the Examples (11b, 11d) and Comparative Examples (11a, 11c) of the present application in distilled water.

12 is a graph showing the immune activity of lactic acid bacteria live bacteria according to an embodiment of the present application.

13 is a graph showing the immune activity of lactic acid bacteria according to an embodiment of the present application, the production of cytotoxicity (LDH) and nitric oxide (Nitric oxide) using a RAW264.7 cell line, a type of macrophage (macrophage) It is a graph measuring the degree.

14 is a graph showing the immune activity of the lactic acid bacteria bacterium according to an embodiment of the present application, cytotoxicity (LDH) and TNF-α, INF-γ, IL-12, IL- using mouse splenocytes. This is a graph measuring the generation of 4.

15 is a photograph and graph showing the anti-obesity effect of the lactic acid bacteria bacteria according to an embodiment of the present application, a photograph showing that the fat accumulation in the adipocytes tested in inverse proportion to the increase in the concentration of the treated lactic acid bacteria bacteria And graph.

DETAILED DESCRIPTION Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present disclosure. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted for simplicity of explanation, and like reference numerals designate like parts throughout the specification.

Throughout this specification, when a part is said to be "connected" with another part, this includes not only the "directly connected" but also the "electrically connected" between other elements in between. do.

Throughout this specification, when a member is located “on” another member, this includes not only when one member is in contact with another member but also when another member exists between the two members.

Throughout this specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding the other components unless otherwise stated. As used throughout this specification, the terms “about”, “substantially”, and the like, are used at, or in close proximity to, numerical values when manufacturing and material tolerances inherent in the meanings indicated are provided, and an understanding of the present application may occur. Accurate or absolute figures are used to assist in the prevention of unfair use by unscrupulous infringers. As used throughout this specification, the term “step of” or “step of” does not mean “step for”.

Throughout this specification, the term "combination (s) thereof" included in the expression of a makushi form refers to one or more mixtures or combinations selected from the group consisting of components described in the expression of makushi form, It means to include one or more selected from the group consisting of the above components.

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

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

Throughout this specification, the description of "culture medium" means "medium containing lactic acid bacteria," "medium inoculated with lactic acid bacteria," "medium containing lactic acid bacteria," "cultivated and concentrated using a membrane filter Medium containing lactic acid bacteria "," medium containing lactic acid bacteria concentrated, dispersed and washed using a membrane filter ", but is not limited thereto.

Throughout this specification, the "size of lactic acid bacteria" refers to the size of one lactic acid bacteria or one individual, and the specific measuring method is different depending on the type and form of lactic acid bacteria. When the lactic acid bacteria are bacillus, it does not mean the length of the bacillus, but means the thickness or width of the bacillus. When the lactic acid bacteria are cocci, it means the diameter of the cocci. In the case of other lactic acid bacteria such as lactic acid bacteria, it means the size measured on the basis of the size commonly used by those skilled in the art.

The first aspect of the present application provides a method for controlling the size of lactic acid bacteria comprising passing the lactic acid bacteria culture medium through a membrane filter.

In one embodiment of the present application, the size of the lactic acid bacteria is to be controlled by the shear force applied to the lactic acid bacteria culture medium, the shear force may be controlled by the speed through which the lactic acid bacteria culture medium passes through the membrane filter.

In one embodiment of the present application, the lactic acid bacteria may comprise lactic acid bacteria live; The lactic acid bacteria culture medium, may comprise a medium inoculated with lactic acid bacteria including lactic acid bacteria live bacteria; The membrane filter may include a hollow fiber membrane filter including a pore having a diameter of 0.1 ㎛ to 1 ㎛.

In one embodiment of the present application, the size of the lactic acid bacteria can be adjusted so that the lactic acid bacteria live bacteria to include lactic acid bacteria having a size of 4.0 ㎛ or less.

In one embodiment of the present application, the lactic acid bacteria comprises lactic acid bacteria; The lactic acid bacteria culture medium is to include a medium containing lactic acid bacteria containing lactic acid bacteria; The membrane filter may include a hollow fiber membrane filter including a pore having a diameter of 0.01 ㎛ to 0.1 ㎛.

In one embodiment of the present application, including the drying and powdering the lactic acid bacteria bacteria, the size of the lactic acid bacteria bacteria is adjusted so that the size of 60% to 100% of the powdered lactic acid bacteria bacteria contains 1.0 μm or less It may be.

In one embodiment of the present application, the lactic acid bacteria may include those selected from Bacillus, cocci, Bifidobacteria and combinations thereof.

In one embodiment of the present application, the lactic acid bacteria may include Lactobacillus plantarum .

The second aspect of the present application provides a method for producing lactic acid bacteria, including a method for adjusting the size of the lactic acid bacteria.

The third aspect of the present application provides a lactic acid bacterium produced by the production method of the lactic acid bacteria.

The fourth aspect of the present application provides an additive containing the lactic acid bacteria.

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

In one embodiment of the present application, the membrane filter may be included in a bioreactor, the bioreactor may comprise a incubator and a membrane filter.

In one embodiment of the present application, the membrane filter includes a first membrane filter and a second membrane filter, thereby applying a shear force to the lactic acid bacteria live bacteria passing through the first membrane filter and the lactic acid bacteria bacteria passing through the second membrane filter. .

Size control method according to an embodiment of the present application by easily applying the shear force to the lactic acid bacteria live bacteria and lactic acid bacteria, the size of the lactic acid bacteria can be easily adjusted, in particular the size of the lactic acid bacteria can be adjusted small. Accordingly, lactic acid bacteria prepared using the size control method according to an embodiment of the present application may not only be effectively absorbed into the body, but also effectively absorbed into the body, thereby exhibiting an immune activity and an 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 the following equation (1).

[Equation 1]

In Equation 1, j (the shear force of the fluid flowing along the tube), m (the viscosity of the fluid), Q (flow velocity of the fluid), r (radius of the tube) and L (length of the tube). In addition, the flow rate of the fluid during the cultivation may act as an important factor to control the shear force.

In one embodiment of the present application, the bioreactor including the incubator and the membrane filter may preferably include the incubator, the first membrane filter and the second membrane filter. Here, the bioreactor may include a structure in which the fluid added to the incubator is independently passed through the first membrane filter and the second membrane filter, respectively, and then delivered to the incubator. FIG. 2A is a schematic diagram of a bioreactor in which the membrane filter ⑥ includes both the first membrane filter and the second membrane filter, and after the fluid is passed through the first membrane filter to the incubator The fluid may be delivered to the incubator through a second membrane filter. In one embodiment of the present application, the first membrane filter and the second membrane filter, unlike in Figure 2 (a) may be present in a space independent of each other.

Furthermore, the bioreactor may further include a tank for producing a medium and a membrane filter for producing a medium. Here, the medium prepared in the medium production tank may be passed to the medium production tank again through the membrane filter for medium production or may be delivered to the incubator. When the medium passing through the membrane filter for producing the medium is delivered to the incubator, the medium may be directly passed to the incubator through the membrane filter for producing the medium. In addition, as shown in (a) of FIG. 2, after the medium is stored in the storage tank ③ before being delivered to the incubator, it may be delivered to the incubator.

By passing the medium inoculated with the lactic acid bacteria live bacteria through the first membrane filter, the lactic acid bacteria live bacteria can be cultured and concentrated. Then, the lactic acid bacteria bacteria can be concentrated and dispersed by passing the medium containing the lactic acid bacteria bacteria produced by killing the cultured and concentrated lactic acid bacteria living bacteria through the second membrane filter.

 In one embodiment of the present application, the first membrane filter may be a hollow fiber membrane filter including a pore having a diameter of the size of the lactic acid bacteria bacteria are not filtered while filtering the impurities contained in the medium inoculated with the lactic acid bacteria bacteria. The pore diameter of the first membrane filter is, for example, 0.1 μm to 1.0 μm, 0.2 μm to 1.0 μm, 0.3 μm to 1.0 μm, 0.4 μm to 1.0 μm, 0.5 μm to 1.0 μm, 0.1 μm to 0.9 Μm, 0.1 μm to 0.8 μm, 0.1 μm to 0.7 μm, 0.1 μm to 0.6 μm or 0.1 μm to 0.5 μm. When the size of the pore diameter of the first membrane filter is less than 0.1 ㎛, severe fouling phenomenon occurs during the culture of the lactic acid bacteria live bacteria, the flux of the culture solution is reduced. Accordingly, a problem may occur that the concentration efficiency of the lactic acid bacteria live bacteria is reduced. When the pore diameter of the first membrane filter is larger than 1.0 μm, the lactic acid bacteria may be filtered through the membrane together with impurities.

In one embodiment of the present invention, the second membrane filter is a hollow fiber membrane comprising a pore having a diameter of the size that can concentrate the lactic acid bacteria reduced in size during the cultivation and concentration of lactic acid bacteria and / or sterilization process It may be a filter. The pore diameter of the second membrane filter is, for example, 0.01 μm to 0.1 μm, 0.02 μm to 0.1 μm, 0.03 μm to 0.1 μm, 0.04 μm to 0.1 μm, 0.05 μm to 0.1 μm, 0.01 μm to 0.09 Μm, 0.01 μm to 0.08 μm, 0.01 μm to 0.07 μm, 0.01 μm to 0.06 μm or 0.01 μm to 0.05 μm . When the size of the pore diameter of the second membrane filter is less than 0.01 ㎛ , severe fouling phenomenon occurs during the concentration of lactic acid bacteria, and the flux of the culture solution is reduced. Accordingly, a problem may occur in which the concentration efficiency of the lactic acid bacteria is reduced. When the pore diameter of the second membrane filter is larger than 0.1 μm , the lactic acid bacteria may be filtered through the membrane together with impurities.

In one embodiment of the present application, 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, according to Equation 1, it is possible to increase the shear force applied to the lactic acid bacteria live bacteria or dead bacteria. Accordingly, the size of the lactic acid bacteria live bacteria or dead bacteria can be adjusted small, and the degree of dispersion can be increased.

In one embodiment of the present application, 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 present disclosure, the bioreactor may include a pressure gauge and / or a flow meter. Here, the pressure gauge is to manage the fouling (fouling) phenomenon of the membrane filter, the flow meter is to measure the flow rate ( Q ) of the culture fluid through the membrane filter in real time to control the shear force applied to the lactic acid bacteria It is for.

In one embodiment of the present application, the bioreactor may further include a tank for producing a medium and a membrane filter for producing a medium. The medium serves to supply nutrients necessary for the culture of lactic acid bacteria live bacteria, can be prepared by sterilizing the medium solution. Conventional medium is usually sterilized by steam, etc., by the steam sterilization, carbohydrates, proteins, etc. are generated carbonized by heat and remain in the medium, the quality of lactic acid bacteria powder may be degraded have.

In one embodiment of the present application, when the bioreactor includes a tank for producing a medium and a membrane filter for producing a medium, the impurities by carbonization in the medium solution are filtered by filtering impurities contained in the medium solution using the membrane filter for producing a medium. Can be prevented. Accordingly, the manufacturing method according to an embodiment of the present application can provide a high quality lactic acid bacteria raw material. Herein, the membrane filter for preparing the medium may include a hollow fiber membrane filter including a diameter of a size capable of filtering impurities contained in the medium solution. The diameter may be 0.1 to 1 μm, but is not limited thereto.

In one embodiment of the present application, the lactic acid bacteria may include those selected from the group consisting of bacilli, cocci, Bifido bacteria 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 Bacillus and Lactococcus lactis , Lactococcus lactis subsp . lactis , Streptococcus thermophilus , Enterococcus aureus, such as faecium , Enterococcus facalis , and Bifidobacterium longum , B. lactis , B. lactis subsp . latis, B. infantis, B. breve, B. there may be mentioned a bipyridinium dogyun of aldolescence, and the like.

In one embodiment of the present application, when the lactic acid bacteria include L. plantarum (LM1001, KCCM accession number 42959) or L. plantarum (LM1004, KCCM accession No. 43246), the effects of the present application may be more marked. .

In one embodiment of the present application, the lactic acid bacteria live bacteria inoculated into the medium may be cultured through one or more culture steps. For example, the lactic acid bacteria live bacteria may be cultured through the step of species culture and intermediate culture.

In one embodiment of the present application, the seed culture is preferably inoculated in a liquid medium after lactic acid bacteria live, and then at a temperature of 20 ℃ to 40 ℃ preferably for 10 to 40 hours, but is not limited thereto. Here, depending on the type of lactic acid bacteria can also be cultured in aerobic conditions.

In one embodiment of the present application, the liquid culture of the species culture may be adjusted to pH 4.0 to pH 8.0 before sterilization using an alkaline solution prepared in advance. The composition (w / v%) of the culture medium is 2.0 to 10.0 w / v% hydrous glucose, 0.1 to 5.0 w / v% soy proteinase, 0.1 to 5.0 w / v% caseinase, 0.1 to 5.0 w / v% yeast extract, 0.01-3.0 w / v% potassium diphosphate, 0.1-5.0 w / v% magnesium sulfate, 0.01-1.0 w / v% calcium chloride, 0.01-0.1 w / v% manganese sulfate, 0.01 To 5.0 w / v% sodium acetate, but is not limited thereto.

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

In one embodiment of the present application, the medium culture liquid medium may be adjusted to pH 4.0 to pH 8.0 prior to sterilization using an alkaline solution prepared in advance. The composition (w / v%) of the intermediate culture medium was 2.0 to 10.0 w / v% hydrous glucose, 0.1 to 5.0 w / v% soy proteinase, 0.1 to 3.0 w / v% L-cysteine, 0.1 to 5.0 w / v% yeast extract, 0.01-3.0 w / v% potassium diphosphate, 0.1-5.0 w / v% magnesium sulfate, 0.01-0.1 w / v% manganese sulfate, 0.01-5.0 w / v% potassium citrate, 0.01 To 2.0 w / v% calcium chloride, 0.01 to 5.0 w / v% surfactant, but is not limited thereto.

In one embodiment of the present application, after the inoculation of the lactic acid bacteria live bacteria cultured through the at least one culture step or the lactic acid bacteria live bacteria not passed through the at least one culture step in the medium, the medium inoculated with the lactic acid bacteria live bacteria in the incubator Add. Subsequently, the lactic acid bacteria live bacteria can be inoculated and concentrated while passing through the first membrane filter inoculated with the lactic acid bacteria live bacteria.

In one embodiment of the present application, the medium in which the lactic acid bacteria live bacteria are inoculated is prepared from the medium solution, the medium is to be produced by sterilization by filtering impurities or contaminants contained in the medium solution through the medium membrane filter Can be. Conventional medium is usually sterilized with steam, etc., by the sterilization of the steam, such as carbohydrates, proteins, such as carbonized impurities are generated and remain in the medium, the quality of lactic acid bacteria powder may be degraded. In one embodiment of the present application, in the process of sterilizing the medium through the medium membrane filter, since the impurities derived from medium, carbonization, etc. generated by conventional steam sterilization does not occur, it is possible to provide a high quality lactic acid bacteria raw material.

Here, the medium-derived impurities may be, but are not limited to, non-water soluble proteins denatured by heat, vitamins or nutrients destroyed by heat, and glucose components decomposed or changed by heat.

The medium to which the lactic acid bacteria are inoculated is sufficient as long as the lactic acid bacteria can be grown and / or cultured, for example, 1.0 to 10.0 w / v% hydrous glucose, 0.1 to 5.0 w / v% soy proteinase. , 0.1-5.0 w / v% yeast extract, 0.01-3.0 w / v% potassium diphosphate, 0.1-5.0 w / v% magnesium sulfate, 0.01-0.1 w / v% manganese sulfate, 0.01-2.0 w / v% It may include calcium chloride, or 0.1 to 10.0 w / v% whey powder and 0.1 to 10.0 w / v% chicory extract, but is not limited thereto. Furthermore, 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 application, the size of the lactic acid bacteria live bacteria may be controlled by the shear force applied to the medium while the medium inoculated with the lactic acid bacteria bacteria passed through the first membrane filter. The shear force may be controlled by the flow rate of the medium inoculated with lactic acid bacteria live bacteria. The shear force can be controlled by the channel size (radius) inside the hollow fiber membrane of the first membrane filter through which the medium inoculated with the lactic acid bacteria live bacteria, the viscosity and the flow rate of the medium (see Equation 1).

According to the conventional batch lactic acid bacteria culture method, there is a problem that it is not easy to adjust the size of the lactic acid bacteria small. In order to solve this problem, the present application by applying a shear force to the medium containing the lactic acid bacteria to stress the lactic acid bacteria in culture, it is possible to easily control the size of the lactic acid bacteria.

It is known that the size of the lactic acid bacteria directly affects the absorption rate of the lactic acid bacteria, and in particular, the smaller the size of the lactic acid bacteria is, the higher the absorption rate of the lactic acid bacteria is and the biological efficacy is increased.

According to the size control method of the present application, as shown in Equation 1, by controlling the flow rate of the medium passing through the first membrane filter, the shear force applied to the medium can be adjusted. Accordingly, the size of the lactic acid bacteria live bacteria to be cultured can be easily adjusted. Preferably, by adjusting the size of the live bacteria of the lactic acid bacteria to 1.0 ㎛ or less, it is possible to maximize the absorption in the body. As described above, the lactic acid bacteria living bacteria, which are relatively small in size, are less aggregated in the subsequent sterilization process, and their sizes are relatively small, so that absorption in the body can be maximized.

In one embodiment of the present application, 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 present application, by passing the lactic acid bacteria live bacteria through the first membrane filter, the size of the lactic acid bacteria living bacteria can be adjusted. Furthermore, impurities in the medium can be continuously removed, and lactic acid bacteria can be concentrated.

In the normal batch lactic acid bacteria culture process, impurities such as proteins, carbohydrates, organic acids, and cell lysates accumulate in the medium inoculated with the lactic acid bacteria, and the lactic acid bacteria are attacked to limit the growth of lactic acid bacteria, thereby lowering the final concentration of lactic acid bacteria. This will cause problems with floating.

However, according to the production method of the present application, such impurities may be filtered through the first membrane filter, thereby exhibiting an effect of increasing the concentration of lactic acid bacteria or live bacteria. Furthermore, since such impurities are filtered, it is possible to simplify the washing process in the recovery process of lactic acid bacteria, it can also exhibit the effect of cost reduction and process simplification.

In one embodiment of the present application, the medium in which the lactic acid bacteria live cells are inoculated may be maintained at pH 5.5 to 6.8 during the culture of the lactic acid bacteria live bacteria. Optimum pH of the medium inoculated with the lactic acid bacteria live bacteria may vary depending on the type of lactic acid bacteria. In one embodiment of the present application, when the organic acid produced by the lactic acid bacteria during the culturing lowers the pH of the medium, a substance that maintains a constant pH may be added to the medium. The material for maintaining the pH of the medium is, for example, sodium hydroxide (NaOH) solution, potassium hydroxide (KOH) solution, ammonia water or ammonia gas, but is not limited thereto.

In one embodiment of the present application, lactic acid bacteria can be made by killing the lactic acid bacteria living cultured and concentrated as described above. The lactic acid bacteria live bacteria may be sterilized by tines or heat treatment, but is not limited thereto.

In one embodiment of the present application, the heat treatment may be performed for 3 to 15 minutes at a temperature of 80 ℃ to 121 ℃. In addition, the heat treatment may be performed by ultra high temperature sterilization 1 to 10 times. The ultra high temperature sterilization may be progressed for 3.0 seconds to 10.0 seconds at a temperature of 110 ℃ to 130 ℃, for example, two times for 1.0 seconds to 10 seconds at 100 ℃, once for 1.0 to 10.0 seconds at 121 ℃ It may be going.

In one embodiment of the present application, the production method of lactic acid bacteria bacterium may further comprise 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 have an effect of preventing agglomeration which may occur during the sterilization process of the lactic acid bacteria live bacteria.

The dispersant may be added to 10.0 (w / w%) to 80.0 (w / w%) with respect to the pellet of the cultured and concentrated lactic acid bacteria. The dispersant may be maltodextrin or trehalose, but is not limited thereto.

In one embodiment of the present application, the lactic acid bacterium bacterium can be concentrated while passing through the second membrane filter a medium containing the sterilized lactic acid bacterium as described above. When the medium containing the lactic acid bacteria bacterium passes through the second membrane filter, the lactic acid bacteria bacterium, particularly, the lactic acid bacteria bacterium aggregated during the killing process of the lactic acid bacteria, may be exhibited by using the shear force applied to the medium. have. Further, by filtering the impurities contained in the medium, it can also exhibit the effect of washing the lactic acid bacteria.

In one embodiment of the present application, passing the medium containing the lactic acid bacteria bacteria through the second membrane filter, it is possible to maximize the washing and concentration of the lactic acid bacteria bacteria. According to this preparation method, the concentration of lactic acid bacteria can be preferably concentrated about 10 times, 9 times, 8 times, 7 times, 6 times or 5 times.

Then, after the lactic acid bacteria are washed, may be dried and powdered. Lactic acid bacteria powder prepared using the size control method of the present application may exhibit an effect of not causing precipitation phenomenon in the solvent. By the production method of the present application, the lactic acid bacteria live bacteria can be adjusted in size by the shear force while passing through the first membrane filter. The size-controlled lactic acid bacteria live bacteria may be aggregated in the process of killing and forming agglomerates, but the lactic acid bacteria bacteria may be dispersed by shear force while passing through the second membrane filter. Therefore, even if the dispersed lactic acid bacteria killed powdered lactic acid bacteria agglomeration phenomenon does not occur, it is possible to prevent the phenomenon of precipitation in the solvent.

 In one embodiment of the present application, the lactic acid bacteria culture solution may be washed to filter impurities such as proteins, carbohydrates, lactic acid bacteria crushed products, except lactic acid bacteria.

In one embodiment of the present application, in order to maximize the effect of the lactic acid bacteria wash by the second membrane filter, after adding the wash solution to the incubator may be passed through the second membrane filter. Here, the washing liquid, for example, sterilized distilled water and distilled water containing 0.1 to 1.0 w / v% sodium chloride (NaCl) and the like, but is not limited thereto. The washing solution may be added at 1.0 to 10 times the volume of the medium containing the concentrated lactic acid bacteria.

In one embodiment of the present application, the drying may be lyophilization, fluidized bed drying or spray drying, but is not limited thereto.

In one embodiment of the present application, the powdered lactic acid bacteria may include a size of 0.01 ㎛ to 3.0 ㎛, 0.1 ㎛ to 3.0 ㎛, 0.5 ㎛ to 3.0 ㎛. Here, the size of 40% to 100% of the powdered lactic acid bacteria may be 1.0 μm or less.

The raw lactic acid bacteria bacterium produced using the size control method of the present application not only has a small particle size of lactic acid bacteria, but is evenly distributed and distributed. Furthermore, since a relatively small amount of impurities are present through the impurity filtration step by the first membrane filter and the second membrane filter, the production of lactic acid bacteria by using the size control method according to the present application may provide high-quality lactic acid bacteria raw material. Can be.

The high-quality lactic acid bacterium bacterium powder of high quality does not cause precipitation when added to the food, functional food or feed, and thus may exhibit an excellent circulation and shelf life.

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

EXAMPLE

Origin of Lactic Acid Bacteria

L.plantarum (LM1001) and L.plantarum (LM1004), as used herein, were separated from Kimchi, a Korean traditional fermented food, at Korea International University Department of Pharmaceutical Engineering (address: 660-759, 965-dong, Munsan-eup, Jinju-si, Gyeongnam). The L.plantarum (LM1001) and L.plantarum (LM1004) were deposited in the Korean culture center of microorganisms in general, and new KCCM 42959 (deposited date: November 12, 2010) and KCCM 43246, respectively. (Deposit date: October 28, 2016).

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

2. Analysis method

2-1. Viable count  analysis

The number of live bacteria of lactic acid bacteria increased during the culture was analyzed by colony counting method. At this time, the medium used to analyze colonies of L. plantarum was MRS (Difco, USA) solid medium, the culture was analyzed by measuring the number of colonies generated after aerobic 48 hours at 37 ℃. The incubator used the JRS-150C model used previously.

The culture solution was diluted with peptone water up to 10 ⁷ , 10 ⁸ , 10 ⁹ and then analyzed using a Pouring method while using 1.0 ml per Petri dish.

The composition (w / v%) of peptone water used for media dilution is as follows: 0.01 to 1.0 sodium chloride, 0.1 to 5.0 w / v% caseinase digest, 0.01 to 5.0 w / v% sodium phosphate, 0.01 to 1.0 w / v% potassium monophosphate was prepared and used after sterilization (121 ° C., 15 minutes).

2-2. Total bacteria  analysis

After sterilization of the culture, the number of microorganisms in the culture was analyzed using a hemocytometer Hemocytometer [Marienfeld, Germany]. The average number N _avg was measured by measuring the number of dead bacteria in 20 cells of the hemocytometer using an optical microscope [BX 53F, Olympus, Japan] at 1,000 magnification. ] Was analyzed.

[Equation 2]

Where R is the dilution factor.

For analysis of the total number of bacteria present in the dead bacterium, 1.0 g dry raw material was dissolved in 10 ml of distilled water, and then suspended and diluted in sterilized distilled water at 10 ⁸ , 10 ⁹ , 10 ¹⁰ .

2-3. Glucose, lactic acid HPLC  analysis

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

2-4. Immune activity assay

Immune activity and cytotoxicity of Heat-killed probiotics (HK-probiotics) were determined by in-vitro assay using splenocytes in macrophages (RAW 264.7) and mouse spleen. 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 Was evaluated by analyzing.

2-4-1. Sample acquisition

L. plantarum (LM1001) was used to prepare lactic acid bacteria live bacteria and lactic acid bacteria dead bacteria samples prepared by the method of the present application.

Lactobacillus probiotic samples were analyzed by in-vivo test to measure the changes of cytokines in the blood of mice by oral administration of diluted live bacteria at a constant concentration. The immunological activity of the bacterium was measured in mouse splenocytes and macrophages (RAW264.7). ) Was carried out through in-vitro experiments to analyze changes in cytokines after treatment for each concentration by killing bacteria.

2-4-2. Laboratory Animals and Processing

Six-week-old male Balb / c mice were used for sale at Samtaco Korea (Korea). Breeding environment was raised under the temperature of 22 ± 2 ℃, humidity 50 ± 20%, lighting for 12 hours, the feed was fed solid feed for mice, the drinking water was unlimited drinking water. The animals were acclimated in the experimental animal room for one week before performing the experiment.

Lactic acid bacteria live bacteria of L. plantarum (LM1001) prepared by the manufacturing method of the present application was carried out 1 oral administration daily for 10 days by concentration. 24 hours after the last oral administration, blood samples were obtained from the experimental animals and stored at -80 ° C for use in the experiment. As a positive control, Con A was administered at 25 mg / kg.

2-4-3. mouse Splenocytes  Isolation and Cultivation

Spleens are aseptically extracted from mice, washed with RPMI 1640 solution and then ground to liberate cells. The separated cell suspension was passed through a 200 mesh stainless steel sieve and then centrifuged at 4 ° C. at 1,200 rpm for 3 minutes to suspend cell pellets in ACK buffer for 5 minutes to remove red blood cells. Outflowed splenocytes were suspended in RPMI 1640 containing 10% fetal bovine serum and 1% penicillin-streptomycin, so that the concentration of cells was 1 × 10 ⁶ cells / ml, and 500 μl was dispensed into 48 well plates. Lactobacillus bacteria of L. plantarum prepared by the method of the present invention were treated by concentration. Lipopolysaccharide and Con A were sampled into positive controls and treated with cells.

Cell groups treated as above were incubated for 3 to 72 hours in a 37 ℃, 5% CO ₂ incubator, the supernatant of the culture was stored at -20 ℃ was used for cytokine production measurement.

2-4-4. Cell culture

Splenocytes and RAW 264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 units / mL of streptomycin and penicillin at 37 ° C., 5% CO ₂ , and their effects on NO and cell proliferation. It was used to find out.

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

After culturing splenocytes, the secretion of cytokines (TNF-α, IFN-γ, IL-12) secreted from the culture supernatant was measured. After treatment with L.plantarum (LM1001) killing bacteria samples prepared by the method of the present invention together with the positive control group LPS and Con A for a predetermined time, the concentration of free satocaine in the culture was measured by enzyme linked immumosorbent assay, Measurement was performed using ELISA (ELISA kit, R & D system, USA).

That is, 100 μl of the supernatant sample was added to a 96 well plate coated with cytokine antibody and allowed to react at room temperature for 2 hours. Then, the supernatant was removed and washed three times with a phosphate round solution and Tween-20 (Sigma). It was washed over.

Again, the solution containing the secondary antibody was added and reacted with the primary antibody, and horseradish peroxidase (HRP) enzyme coupled with avidin was added and reacted at room temperature for 15 minutes. Thereafter, the reaction was confirmed by changing the color by putting the TMB solution as a substrate for the HRP enzyme.

Since the change of color appears depending on the concentration of cytokines present in the sample, it was possible to know whether or not cytokines were produced in the sample. After the reaction of the HRP enzyme with the TMB substrate was terminated by adding sulfuric acid (1.0 M), the absorbance was measured at 450 nm using a microplate reader (Thermo).

2-4-6. NO measurement

Macrophage lines RAW 264.7 cells were aliquoted into 96 well plates at a concentration of 5 × 10 ⁵ cells / ml and incubated for 24 hours, and then treated with 1 mg / ml of LPS as a positive control and phosphate buffer solution as a negative control. The L.plantarum (LM1001) bacterium sample prepared by the method of the present invention was treated and then incubated again for 24 hours.

The concentration of NO was measured using a Greases reagent system (Sigma, USA). 50 μl culture solution was added to a 96-well plate, and grease Reagent I (NED solution) and Grease Reagent II (sulfanylamide solution) were mixed in the same amount, and then reacted in a dark room for 10 minutes and within 30 minutes, a microplate reader (Tecan, Austria) at 540 nm. The concentration of NO was calculated using a standard curve of sodium nitrite (0-100 micromol).

2-4-7. Cell proliferation rate measurement

Proliferation of splenocytes and RAW 264.7 cells was determined by WST-1 assay. Splenocytes and RAW 264.7 cells were adjusted to a concentration of 5 × 10 ⁵ cells / well, and the cells were inoculated into 96-well plates and treated with samples and controls for 48 hours. 100 μl of WST-1 kit solution was added and incubated for 1 hour, and the absorbance was measured at 540 nm using an ELISA reader (Thermo, Germany).

[Equation 3]

Here, N (%) is the cell growth ratio, C _p is the absorbance after killing, C _s is the absorbance after no treatment.

2-4-8. Statistical processing

The results obtained in the above experiments were analyzed by a one way ANOVA test using the SPSS 21 manual (Statistical Package for Social Sciences, IBM, Armonk, NY, USA), and the significance between samples was Student to Tail. The T test was used to compare at the P <0.05 level.

2-5. Lactobacillus anti-obesity effect analysis

Induction of differentiation of fat precursor cells (3T3-L1 cells) into mature adipocytes, after treatment with different concentrations of lactic acid bacteria prepared according to the embodiment by concentration, oil red O (Oil red to confirm the effect of inhibiting fat accumulation in fat cells O) staining and intracellular triglyceride content (TG kit) was measured to determine the degree of intracellular fat accumulation.

2-5-1. Fat precursor cell  Growth inhibition effect

In order to analyze the anti-obesity effect of lactic acid bacteria, MTT assay (Methylthiazolinlinyldiphenyl tetrazolium bromide assay) was performed to analyze the cytotoxicity of lactic acid bacteria microorganisms in the culture medium of progenitor cells 3T3-L1 (Mouse Embryonic Fibroblast-Adipose like cell line).

3T3-L1 adipocytes were aliquoted into 4 × 10 ⁴ cells / ml in 96-well plates (96-well plates) and incubated in a 5% CO ₂ , 37 ° C. incubator for 24 hours. After incubation, the culture medium was removed, and DMEM medium and lactic acid bacteria microbial powder were treated by concentration (0, 100, 250, 500 µg / ml) for 24 hours. Then, MTT (3- [4,5-dimethyl-thiazol] -2,5-diphenyl-tetrazolium bromide) dissolved in 5 mg / ml in solvent DPBS (Dulbecco's Phosphate-Buffered Saline, 1X) was added to each well. 120 μl of each was incubated in a 5% CO ₂ , 37 ° C. incubator for 4 hours.

After incubation, the culture medium was removed, and each well was mixed with a pipette while adding dimethyl sulfoxide (DMSO; dimethyl sulfoxide) at 200 μl / well. Thereafter, the absorbance was measured at 570 nm with an ELISA reader.

2-5-2. Adipocyte lipid accumulation inhibitory activity (Oil Red O staining analysis)

Dispense 3T3-L1 adipocytes into 6-well plates (6-well plates) at a density of 7 × 10 ⁴ cells / ml and differentiate the adipocytes for 8 days while simultaneously adding lactic acid bacterium powder by concentration (100, 250, 500 μg). / ml). After 8 days, the culture medium was discarded and washed with phosphate buffered saline (PBS). Thereafter, 10% formaldehyde (formaldehyde) in each well was added to each well of 500 μl and fixed at 4 ° C. for 1 hour. After removing formaldehyde, washed three times with phosphate buffered saline and dried in a CO ₂ incubator.

Then, when the plate was dried, the oil red o dye was added 500 ㎕ each dye for 30 minutes in a dark state at room temperature and washed three times with phosphate buffered saline. The stained cells were observed under a microscope, and after observation, the dyes stained in adipocytes were extracted with 300 μl of iso-propanol per well, and an optical density (OD value) was measured at 500 nm with an ELISA reader.

Oil Red O Dye was used after mixing 500 mg of Oil Red O Dye in 100 ml of iso-propanol, mixed with distilled water at a ratio of 6: 4, and filtering with a 0.45 μm filter.

2-5-3. Triglycerides in fat cells ( Tri - glycerides A) content analysis

Dispense 3T3-L1 adipocytes into 6-well plates (6-well plates) at a density of 7 × 10 ⁴ cells / ml and differentiate the adipocytes for 8 days while simultaneously adding lactic acid bacterium powder by concentration (100, 250, 500). Μg / ml).

After 8 days, each well was washed with phosphate buffered saline, and the cells were collected using a scraper (cell scraper, SPL), and triglyceride kit (TG) was extracted by extracting the triglycerides in the cells using an ultrasonic grinder (SONIFIER 450, BRANSON). -S, Asan Pharmaceutical) was used to measure the content of triglycerides.

Example  1. Cultivation and concentration of live bacteria

1-1. Storage of Lactobacillus Probiotics

Lactic acid bacteria to be used in the Examples herein were first incubated in anaerobic conditions for 48 hours using MRS (Difco. USA) solid medium at 37 ℃. After 48 hours, the lactic acid bacteria community shown in the medium was harvested in a sterile bench and washed three times with phosphate buffered saline (PBS, pH 6.8), followed by appropriate cryoprotectant (25% glycerin + 10% skim milk powder). Suspension was added and suspended 0.2 ml each in Cryo-vial (1.2 ml, Simport, Canada) prior to freezing in a -70 ℃ cryogenic freezer.

The frozen vials (Defreezed vials, DF vials) were stored for up to 6 months, taken out and used as needed when dissolved. All experiments were carried out in aseptic conditions to minimize contamination.

1-2. Lactic acid bacteria Cultivation

The vial was taken out of the cryogenic freezer and dissolved, followed by seed culture of lactic acid bacteria.

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

Secondly, 15 ml lactic acid bacteria cultured for 12 hours were inoculated again with 1.0 L culture medium LTMS-PR-SM (2.0 L Erlenmeyer flask, 4), and the incubator was subjected to aerobic conditions at 37 ° C. for 10 hours. JSRB-150C, JSR Korea].

The species culture medium LTMS-PR-SM composition (w / v%) used was as follows: 2.0-10.0 w / v% hydrous glucose, 0.1-5.0 w / v% soy proteinase, 0.1-5.0 w / v% caseinase digest, 0.1 to 5.0 w / v% yeast extract, 0.01 to 3.0 w / v% potassium diphosphate, 0.1 to 5.0 w / v% magnesium sulfate, 0.01 to 1.0 w / v% calcium chloride, 0.01 to 0.1 w / v% manganese sulfate, 0.01-5.0 w / v% sodium acetate, and the like, adjusted to pH 6.8 with NaOH (10.0 M) prior to sterilization.

1-3. Medium culture of lactic acid bacteria

1.5 L species culture cultured in the above species cultures were used to inoculate 80 L medium culture medium LTMS-PR-MM (100 L fermenter, CobioTack). This intermediate culture was carried out for 12 hours with slight agitation at 32 ° C. and ammonia gas was added periodically as needed to adjust the pH of the culture to pH 5.0 to pH 7.0.

The composition (w / v%) of the intermediate culture medium used at this time is as follows: 2.0 to 10.0 w / v% hydrous glucose, 0.1 to 5.0 w / v% soy proteinase, 0.1 to 3.0 w / v% L-cysteine, 0.1-5.0 w / v% yeast extract, 0.01-3.0 w / v% potassium diphosphate, 0.1-5.0 w / v% magnesium sulfate, 0.01-0.1 w / v% manganese sulfate, 0.01-5.0 w / v% potassium citrate, 0.01-2.0 w / v% calcium chloride, 0.01-5.0 w / v% Tween-80, and adjusted to pH 6.8 with NaOH (10.0 M) prior to sterilization.

1-4. Lactic acid bacteria The first act  Culture and Concentration with Filter

The bioreactor used in the size control method of the present application includes a first membrane filter (a hollow fiber membrane filter having a pore size of 0.5 μm diameter and a 6 mm channel radius, an Al-Al ceramic hollow fiber membrane filter) and an incubator. The filter is located outside of the incubator, and the culture medium is first inoculated into the incubator, and then 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.) Will cycle.

First, 20 L of the medium inoculated with the lactic acid bacteria living cells cultured through the above-mentioned species culture and intermediate culture was inoculated into the incubator. Thereafter, the medium containing the lactic acid bacteria live bacteria was incubated with concentration and filtration by passing through the first membrane filter using a forced aseptic circulation pump.

Through the above concentration and filtration process, while the medium culture medium was filtered, fresh medium was added to the incubator. The composition (w / v%) of the freshly inoculated medium is as follows: 2.0 to 10.0 w / v% hydrous glucose, 0.1 to 5.0 w / v% soy proteinase, 0.1 to 5.0 w / v% yeast extract , 0.01-3.0 w / v% potassium diphosphate, 0.1-5.0 w / v% magnesium sulfate, 0.01-0.1 w / v% manganese sulfate, 0.01-2.0 w / v% calcium sulfide. The freshly inoculated medium was previously sterilized through a medium filter (Al-Al ceramic hollow fiber membrane filter having a pore size of 0.2 μm diameter and a 6 mm channel radius) before being added to the incubator.

After completely replacing the intermediate culture medium with freshly added medium, the incubator was cooled to 18 ° C. or lower, and the flow of the culture liquid flowing along the first membrane filter was kept to a minimum. The pH of the medium was maintained at 5.0 to 6.8 (1: 1 = 20%, w / v aqueous ammonia: distilled water) for 5 hours before starting the culture and concentration of the lactic acid bacteria.

Thereafter, the temperature of the bioreactor is raised to 32 ° C. before starting the culture and concentration of the lactic acid bacteria, and the culture medium containing the medium in which the lactic acid bacteria is inoculated is passed through the first membrane filter, thereby culturing the lactic acid bacteria and Substantial cultivation of concentration was initiated. At this time, the volume of the culture solution is reduced at a constant rate while passing through the first membrane filter. The newly inoculated medium is aseptically added to the incubator by using a pump (YZ15, Leadfluid, China, Max = 20.0L / h). Continued feeding.

In addition, the flow rate of the 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, and ammonia water (1: 1 = 20%, w / v aqueous ammonia: distilled water) was used to maintain a pH of 5.0 to pH 7.0, the internal pressure of the fermenter was maintained at 0.1 to 0.5 kg / cm ² state using nitrogen gas to maintain an aerobic state.

Example  2. of lactic acid bacteria Sterile

In one embodiment of the present application, prior to the sterilization of the lactic acid bacteria microorganisms obtained in Example 1, group 1, without dispersant added to the medium inoculated with the lactic acid bacteria microorganisms and 0.1 to 80.0 w / v% maltodextrin and Groups 2 and 3 were prepared with addition of 0.1-80.0 w / v% of trehalose, respectively.

In one embodiment of the present application, the lactic acid bacteria live bacteria obtained in Example 1 was circulated three times at 121 ℃ 3 seconds using an ultra-high temperature continuous sterilizer, to prepare lactic acid bacteria bacteria (Example 2-1).

In one embodiment of the present application, the lactic acid bacteria live bacteria obtained in Example 1 was circulated for 5 minutes at 110 ℃ using an ultra-high temperature continuous sterilizer, to prepare lactic acid bacteria bacteria (Example 2-2, Figure 5b).

In one embodiment of the present application, the lactic acid bacteria live bacteria obtained in Example 1 was circulated for 3 minutes at 65 ℃, 85 ℃ and 120 ℃ using an ultra high temperature continuous sterilizer, to prepare lactic acid bacteria bacteria (Example 2-3, 7).

In one embodiment of the present application, the lactic acid bacteria live bacteria obtained in Example 1 were circulated at 0, 30, 60, 90 and 120 minutes at 80 ° C., respectively, to prepare lactic acid bacteria (Example 2-4). , FIG. 9).

Example  3. Lactobacillus Sterile 2nd membrane  Concentrate, Wash, and Disperse Using Filters

The second membrane filter (0.01 μm to 0.1 μm in pore size and 3 mm channel radius, and the medium having a molecular weight of 50,000 Daltons to 500,000 Daltons were filtered through the medium containing the lactic acid bacterium obtained in Example 2-1. Lactic acid bacteria were concentrated 2 to 10 times by passing through a polysulfone hollow fiber membrane filter). Using the second membrane filter, lactic acid bacteria were dispersed by applying a shearing force to the lactic acid bacteria culture solution aggregated in the sterilization process of Example 2.

Additionally, in order to maximize the effect of the lactic acid bacteria dispersion by the second membrane filter, a dispersant was added to the medium, and all the results are shown in Table 2 and FIGS. 8 and 9.

As shown in Figure 8, when lactic acid bacteria bacteria are concentrated and washed using a second membrane filter, Lactobacillus aggregated in the heat treatment step was found to be dispersed and concentrated to recover. Furthermore, even in the case of using the second membrane filter, it is obvious to those skilled in the art that impurities are filtered and removed from the culture as in the first membrane filter.

As described above, lactic acid bacteria are aggregated in the process of killing the lactic acid bacteria to form a lump of lactic acid bacteria. In this embodiment, the size of the lactic acid bacteria killing bacteria formed immediately after the killing of lactic acid bacteria is 20 ㎛ To 200 μm (FIG. 9). Lactobacillus bacteria or Lactobacillus bacteria bacteria immediately after the sterile bacteria were passed through the second membrane filter to disperse the lactic acid bacteria bacteria bacteria aggregates. The size of the lactic acid bacterium dispersed in this way was measured to 0.5 ㎛ to 3.0 ㎛ or less (Fig. 9).

Example  4. Lactobacillus Sterile  Washing, drying and Powder

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

After sterilized distilled water was added to twice the volume of the medium containing the concentrated lactic acid bacteria, the process of concentrating the diluted medium to the volume before dilution while passing through the second membrane filter was repeated three times. As a result, the concentrated bacteria were washed with a total of 6 times the volume of sterilized distilled water of the medium containing the concentrated bacteria.

Lactobacillus bacteria washed by the above method was dried and powdered in a freeze dryer for a total of 96 hours.

The size distribution of the washed, dried and powdered lactic acid bacteria as described above is shown in FIG. 9. As shown in FIG. 9, the size of the powdered dead bacteria showed a size distribution of 0.5 μm to 3.0 μm, and furthermore, the size of 60% to 100% of the powdered dead bacteria appeared to be 1.0 μm or less. .

Comparative example  One. Batch  Lactic acid bacteria using culture Sterile  Produce

Batch cultivation of lactic acid bacteria was carried out in a total amount of 80 L culture medium in a 100 L fermentation tank (CobioTack). First, 4.0 L (5.0 w / v%) of a culture solution containing lactic acid bacteria living cultured through the species culture in the fermenter was inoculated into a 100 L fermenter.

During the cultivation in a batch incubator, the stirring speed was maintained at 30 rpm and the temperature at 32 ° C. In addition, the aerobic state was maintained using nitrogen gas, and the pH of the culture was maintained at pH 5.0 to pH 7.0 while using ammonia gas. During the batch culture, the live bacteria of the lactic acid bacteria were sampled at regular intervals and analyzed.

Thereafter, in one embodiment of the present application, the live bacteria of the lactic acid bacteria were heat-treated at 110 ° C. for 5 minutes (Comparative Example 1-1, FIG. 5). As shown in Figure 5, lactic acid bacteria produced using a conventional batch culture was found to be larger and less concentrated than the lactic acid bacteria bacteria according to the present invention.

Then, in one embodiment of the present application, the live bacteria of the lactic acid bacteria were circulated by circulating three times for 3 seconds at 120 ℃ (Comparative Example 1-2, Figure 7). As shown in FIG. 7, the lactic acid bacteria bacterium produced using the conventional batch culture was observed to be larger in size than the lactic acid bacteria bacterium according to the present invention.

In addition, lactic acid bacteria were dried and powdered in the same manner as in Example 4.

[ Experimental Example ]

Experimental Example  1. Evaluation of productivity and growth rate of live bacteria

The productivity and growth rate of the lactic acid bacteria live bacteria according to Example 1 and Comparative Example 1 are shown in Table 1 below. Productivity of the lactic acid bacteria live bacteria is represented by the amount of pellets, the growth rate of the lactic acid bacteria (Specific growth rate, m) is represented by the following equation 4.

[Equation 4]

Here, LN is a logarithmic expression, t ₁ represents the concentration of lactic acid bacteria live bacteria at a specific time t ₁ , t ₂ represents the concentration of lactic acid bacteria living bacteria at a specific time t ₂ , t represents the elapsed time.

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

[Equation 5]

Where Q _h was the amount of medium (L) consumed per hour and V is the total volume (L) of the bioreactor.

As shown in Table 1, the lactic acid bacteria live bacteria according to Comparative Example 1 pellets are 18.3 g / L (LM1001) and 15.2 g / L (LM1004), respectively, the lactic acid bacteria live bacteria according to Example 1 is the amount of pellets It was found that excellent productivity was shown at 205.8 g / L (LM1001) and 170.0 g / L (LM1004), respectively.

In addition, the lactic acid concentration of the culture medium of lactic acid bacteria according to Comparative Example 1 was 478.1 mM (LM1001) and 534.1 mM (LM1004), respectively, whereas the culture medium of lactic acid bacteria of Example 1 was 324.3 mM (LM1001), respectively. ) And 369.2 mM (LM1004).

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

Experimental Example  2. Evaluation of the Control of the Lactobacillus Probiotics

After recovering the lactic acid bacteria microorganisms according to Example 1 and Comparative Example 1 and dry powdered, the photograph of the lactic acid bacteria bacteria present in the powder with a scanning electron microscope (Scanning ElectroMicroscope, SEM, X10,000) is shown in FIG. , A photo taken with an optical microscope (X1,000) is shown in FIG. 5.

In addition, the size distribution of the lactic acid bacteria live bacteria according to Example 1 and Comparative Example 1 was measured and shown in FIG.

As shown in Figure 4 and 5, the lactic acid bacteria live bacteria according to Example 1, it was found that not only the size of the lactic acid bacteria live bacteria, but also more concentrated than the comparative example 1.

Furthermore, as shown in Figure 6, the size of the lactic acid bacteria live bacteria according to Comparative Example 1 showed a distribution having a size of up to 6.3㎛, while the size of the lactic acid bacteria live bacteria according to Example 1 has a smaller size of up to 4.0 ㎛ Distribution. In addition, when the lactic acid bacteria live bacteria according to Example 1 was heat-treated and sterilized, the size was reduced to 3.0 ㎛, which indicates that the value is significantly smaller than 4.0 ㎛ of Comparative Example 1.

Experimental Example  3. Lactobacillus Sterile  Scale rating

In the recovery process of lactic acid bacteria according to Example 2-1, 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.

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

Experimental Example  4. Lactobacillus Sterile Suspension  evaluation

This experiment was conducted to observe the stability of the suspended state of the original lactic acid bacteria bacteria according to Example 4 and Comparative Example 1-1.

The experiment was prepared using the Lactobacillus plantarum (LM1001) lactic acid bacteria, the original lactic acid bacteria according to Example 4 and Comparative Example 1-1 to 1.0E + 12 / g raw powder. Thereafter, the dead bacteria-derived original bacteria of Example 4 were 1.0E + 8 / ml concentration and the dead bacteria-derived original bacteria of Comparative Example 1-1 were suspended in sterilized distilled water at a concentration of 1.0E + 7 / ml for 4 days at room temperature. The precipitation state was observed (FIG. 11).

The total bacterium density of lactic acid bacteria killing bacteria prepared by culturing using the size control method of the present application was 5.2E + 12 / g, and the total bacterial density of lactic acid bacteria killing bacteria prepared by batch culture was 2.9E + 12. / g was the end.

As shown in FIG. 11, when batch culture is used, a part of the suspension is precipitated within one hour of suspension, while the suspension of the present invention is stably maintained for four days after suspension. It can be seen that.

Experimental Example  5. Analysis of immune activity of live bacteria

Immune activity was analyzed for live and dead bacteria of two species of L. plantarum prepared using the size control method of the present application.

The immunological activity of the lactic acid bacteria was analyzed as follows: After feeding the mouse with the feed containing the lactic acid bacteria, the concentration of cytokines (TNF-α, INF-γ, IL-12) in the mouse blood was measured. An in-vivo test was performed to show in FIG. 12.

Specifically, two-week-old male Balb / c mice were orally administered with lactic acid bacteria two species once daily for 10 days at different concentrations (10, 100 mg / kg). 24 hours after the last oral administration, blood samples were obtained from the experimental animals and stored at -80 ° C for use in the experiment. As a positive control, Con A was administered at 25 mg / kg.

As shown in FIG. 12, after treating the lactic acid bacteria for 10 days, the amount of serum cytokines was measured using an ELISA assay. Cytokines TNF-α, IFN-γ, and IL-12 were all significantly increased compared to positive Con A (25 mg / kg).

Experimental Example  6. Lactobacillus Sterile  Immune activity assay

Immune activity was analyzed against two strains of L. plantarum prepared using the size control method of the present application.

Immune activity of the lactic acid bacteria are dead cells was assayed as follows: RAW264.7 cells for toxicity experiments with macrophage and NO ^- (Nitric oxide) generation, and mouse spleen cells (splenocytes) cytokines ((TNF-α, INF by -γ, IL-12, IL-6) was performed in-vitro test to measure the production is shown in FIG.

The cytotoxicity and nitric oxide production-inducing functions of lactic acid bacteria were investigated using RAW264.7 macrophages according to the procedure described in the above method.

Fig. 13 (A) shows the results of experiments on cytotoxicity after treating lactic acid bacteria samples for up to 48 hours in order to set the cell treatment concentrations of lactic acid bacteria in RAW 264.7 cells, which are mouse macrophage lines, at concentrations of 10 μg / ml or less. No cytotoxicity is indicated. Therefore, the efficacy of the immunological activity of the lactic acid bacteria sample was measured at a concentration at which no cytotoxicity was observed (0.1-10 µg / ml or less). Cytotoxicity was measured by the activity of free lactate dehydrogenase.

FIG. 13 (B) shows that lactic acid bacteria samples were treated at 0.1, 1, and 10 ug / ml concentrations for 48 hours in the RAW 264.7 cells in order to evaluate the effect of Lactobacillus pylori powder treatment on the NO production ability in Raw 264.7 cells. After treatment up to the activity of the NO released in the culture medium was measured using the Griess reagent system (Griess reagent system) method is shown.

As shown in FIG. 13, the production capacity of NO was increased by about 6 times by LPS, a positive control, and it was confirmed that the concentrations were significantly increased in all of the treated lactic acid bacteria samples.

Figure 14 shows the effect of lactic acid bacteria bacterium powder in cytokine production using mouse spleen cells. Figure 14 (A) shows that the splenocyte cytotoxicity does not appear below 0.1 to 10mg / ml treatment concentration of the lactic acid bacteria killing bacteria powder described above. 14 (B), (C), (D) and (E) show that the treatment of the lactic acid bacteria killing powder increases the production of cytokines significantly in concentration.

As shown in Figures 12 to 14, the lactic acid bacteria of both species were found to be immune enhancing function in live bacteria and dead bacteria, it can be seen that it exhibits immune activity.

Experimental Example  7. Lactobacillus Sterile  Anti-obesity effect

In order to observe the anti-obesity effect of lactic acid bacteria, fat accumulation inhibition experiment in adipocytes was performed. Specifically, after adding 25 mg / ml con A, a fat accumulation inducer, to the adipocytes , L. planatrum (LM1004) was treated with lactic acid bacterium powder by concentration (0, 100, 250, 500 ㎍ / ml), The amount of reduced fat accumulation was measured. As a control group, a group not treated with the fat accumulation inducer and a group not treated with the lactic acid bacterium powder after treatment with the fat accumulation inducer were used. As shown in FIG. 15, it was observed under a microscope that the amount of lipid accumulated in adipocytes decreased concentration-dependently according to the treatment concentration (0, 100, 250, 500 µg / ml) of the lactic acid bacteria bacterium powder ( 15A).

In particular, at a concentration of 500 ㎍ / ml, compared with the positive control group (Con A), it was confirmed by the oil red o staining method to reduce the lipids in fat cells by about 46% (Fig. 15C).

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

Experimental Example 7 shows that LM1004 has an effect of inhibiting lipid accumulation in adipocytes.

The above description of the present application is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present application. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the above description, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present application.

[Accession number]

Depositary Name: Korea Microorganism Conservation Center

Accession number: KCCM43246

Deposit date: 20161028

Depositary Name: Korea Microorganism Conservation Center

Accession number: KCCM42959

Deposit date: 20101112

Claims (11)

A method for controlling the size of lactic acid bacteria, comprising passing the lactic acid bacteria culture medium through a membrane filter. The method of claim 1, The size of the lactic acid bacteria is controlled by the shear force applied to the lactic acid bacteria culture medium, the shear force is controlled by the speed of the lactic acid bacteria culture medium passes through the membrane filter,  How to control the size of lactic acid bacteria. The method of claim 1, The lactic acid bacteria include lactic acid bacteria live bacteria; The lactic acid bacteria culture medium is one containing a medium inoculated with lactic acid bacteria containing live bacteria; Wherein the membrane filter comprises a hollow fiber membrane filter comprising a pore having a diameter of 0.1 ㎛ to 1 ㎛, How to control the size of lactic acid bacteria. The method of claim 3, wherein The size of the lactic acid bacteria live bacteria is adjusted so that the lactic acid bacteria live bacteria having a size of less than 4.0 ㎛, How to control the size of lactic acid bacteria. The method of claim 1, The lactic acid bacteria include lactic acid bacteria; The lactic acid bacteria culture medium is one containing a medium containing lactic acid bacteria including lactic acid bacteria dead bacteria; Wherein the membrane filter comprises a hollow fiber membrane filter comprising a pore having a diameter of 0.01 ㎛ to 0.1 ㎛, How to control the size of lactic acid bacteria. The method of claim 5, wherein Drying and powdering the lactic acid bacteria, The size of the lactic acid bacteria is adjusted so that the size of 60% to 100% of the powdered lactic acid bacteria, including 1.0 ㎛ or less, How to control the size of lactic acid bacteria. The method of claim 1, The lactic acid bacteria, including those selected from Bacillus, cocci, Bifidobacteria and combinations thereof How to control the size of lactic acid bacteria. The method of claim 1, The lactic acid bacteria will include Lactobacillus plantarum , How to control the size of lactic acid bacteria. Claim 1 to claim 8, including a method for adjusting the size of the lactic acid bacteria according to any one of, lactic acid bacteria production method. Lactic acid bacteria prepared by the method for producing lactic acid bacteria according to claim 9. As an additive containing the lactic acid bacteria of claim 10, The additive may be added to food, functional food or feed, additive.

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