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WO2018101612A1 - Procédé de contrôle de la taille des lactobacilles à l'aide d'un filtre à membrane - Google Patents

Procédé de contrôle de la taille des lactobacilles à l'aide d'un filtre à membrane Download PDF

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Publication number
WO2018101612A1
WO2018101612A1 PCT/KR2017/012087 KR2017012087W WO2018101612A1 WO 2018101612 A1 WO2018101612 A1 WO 2018101612A1 KR 2017012087 W KR2017012087 W KR 2017012087W WO 2018101612 A1 WO2018101612 A1 WO 2018101612A1
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Prior art keywords
lactic acid
acid bacteria
bacteria
size
membrane filter
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Ceased
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PCT/KR2017/012087
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English (en)
Korean (ko)
Inventor
정일선
손민
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Lactomason Co Ltd
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Lactomason Co Ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/332—Promoters of weight control and weight loss
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum

Definitions

  • 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.
  • probiotics specified by the World Health Organization is a live microorganisms that, when administered in adequate amounts, confer a health benefit on the host).
  • WHO World Health Organization
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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.
  • the membrane filter 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.
  • 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.
  • FIG. 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.
  • FIG. 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).
  • FIG. 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. 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.
  • 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.
  • FIG. 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.
  • FIG. 12 is a graph showing the immune activity of lactic acid bacteria live bacteria according to an embodiment of the present application.
  • FIG. 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.
  • FIG. 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- ⁇ , INF- ⁇ , IL-12, IL- using mouse splenocytes. This is a graph measuring the generation of 4.
  • LDH cytotoxicity
  • 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.
  • 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.
  • bioreactor means “the whole conventional system capable of producing microorganisms such as lactic acid bacteria”.
  • 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.
  • 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.
  • the lactic acid bacteria are bacillus, it does not mean the length of the bacillus, but means the thickness or width of the bacillus.
  • 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.
  • 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.
  • 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 ⁇ m to 1 ⁇ m.
  • 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 ⁇ m or less.
  • 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 ⁇ m to 0.1 ⁇ m.
  • 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 ⁇ m or less It may be.
  • the lactic acid bacteria may include those selected from Bacillus, cocci, Bifidobacteria and combinations thereof.
  • 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.
  • the membrane filter may be included in a bioreactor, the bioreactor may comprise a incubator and a membrane filter.
  • 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 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.
  • 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 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).
  • the flow rate of the fluid during the cultivation may act as an important factor to control the shear force.
  • the bioreactor including the incubator and the membrane filter may preferably include the incubator, the first membrane filter and the second membrane filter.
  • 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.
  • the first membrane filter and the second membrane filter unlike in Figure 2 (a) may be present in a space independent of each other.
  • the bioreactor may further include a tank for producing a medium and a membrane filter for producing a medium.
  • 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.
  • 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.
  • the medium may be delivered to the incubator.
  • the lactic acid bacteria live bacteria 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.
  • 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 ⁇ m to 1.0 ⁇ m, 0.2 ⁇ m to 1.0 ⁇ m, 0.3 ⁇ m to 1.0 ⁇ m, 0.4 ⁇ m to 1.0 ⁇ m, 0.5 ⁇ m to 1.0 ⁇ m, 0.1 ⁇ m to 0.9 ⁇ m, 0.1 ⁇ m to 0.8 ⁇ m, 0.1 ⁇ m to 0.7 ⁇ m, 0.1 ⁇ m to 0.6 ⁇ m or 0.1 ⁇ m to 0.5 ⁇ m.
  • the size of the pore diameter of the first membrane filter is less than 0.1 ⁇ m, 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.
  • the pore diameter of the first membrane filter is larger than 1.0 ⁇ m, the lactic acid bacteria may be filtered through the membrane together with impurities.
  • 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 ⁇ m to 0.1 ⁇ m, 0.02 ⁇ m to 0.1 ⁇ m, 0.03 ⁇ m to 0.1 ⁇ m, 0.04 ⁇ m to 0.1 ⁇ m, 0.05 ⁇ m to 0.1 ⁇ m, 0.01 ⁇ m to 0.09 ⁇ m, 0.01 ⁇ m to 0.08 ⁇ m, 0.01 ⁇ m to 0.07 ⁇ m, 0.01 ⁇ m to 0.06 ⁇ m or 0.01 ⁇ m to 0.05 ⁇ m .
  • the size of the pore diameter of the second membrane filter is less than 0.01 ⁇ m , 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.
  • the pore diameter of the second membrane filter is larger than 0.1 ⁇ m , the lactic acid bacteria may be filtered through the membrane together with impurities.
  • 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.
  • 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.
  • 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.
  • the bioreactor may include a pressure gauge and / or a flow meter.
  • 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.
  • 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.
  • the manufacturing method according to an embodiment of the present application can provide a high quality lactic acid bacteria raw material.
  • 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 ⁇ m, but is not limited thereto.
  • the lactic acid bacteria may include those selected from the group consisting of bacilli, cocci, Bifido bacteria and combinations thereof.
  • bacilli cocci
  • Bifido bacteria 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.
  • the effects of the present application may be more marked. .
  • the lactic acid bacteria live bacteria inoculated into the medium may be cultured through one or more culture steps.
  • the lactic acid bacteria live bacteria may be cultured through the step of species culture and intermediate culture.
  • the seed culture is preferably inoculated in a liquid medium after lactic acid bacteria live, and then at a temperature of 20 °C to 40 °C preferably for 10 to 40 hours, but is not limited thereto.
  • lactic acid bacteria can also be cultured in aerobic conditions.
  • 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.
  • 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.
  • 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.
  • the medium inoculated with the lactic acid bacteria live bacteria in the incubator Add.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the size control method of the present application 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 ⁇ m 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the heat treatment may be performed for 3 to 15 minutes at a temperature of 80 °C to 121 °C.
  • 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 °C to 130 °C, for example, two times for 1.0 seconds to 10 seconds at 100 °C, once for 1.0 to 10.0 seconds at 121 °C It may be going.
  • 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.
  • 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.
  • 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.
  • the concentration of lactic acid bacteria can be preferably concentrated about 10 times, 9 times, 8 times, 7 times, 6 times or 5 times.
  • 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.
  • 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.
  • 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.
  • 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.
  • the drying may be lyophilization, fluidized bed drying or spray drying, but is not limited thereto.
  • the powdered lactic acid bacteria may include a size of 0.01 ⁇ m to 3.0 ⁇ m, 0.1 ⁇ m to 3.0 ⁇ m, 0.5 ⁇ m to 3.0 ⁇ m.
  • the size of 40% to 100% of the powdered lactic acid bacteria may be 1.0 ⁇ m 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.
  • L.plantarum (LM1001) and L.plantarum (LM1004) 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).
  • lactic acid bacteria used in the following examples or experimental examples are L.plantarum (LM1001) and L.plantarum (LM1004) unless otherwise noted.
  • the number of live bacteria of lactic acid bacteria increased during the culture was analyzed by colony counting method.
  • 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 °C.
  • 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.
  • 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).
  • 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.
  • R is the dilution factor
  • HPLC Agilent Technology 1200, Agilent, USA
  • Amine-column NH 2, 300mm x 7.8mm, 9.0mm particle size, Bio-Rad, USA
  • 5mM H 2 SO 4 solution 0.5ml flow rate , respectively
  • 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.
  • HK-probiotics Heat-killed probiotics
  • IL-4 interleukin -4
  • INF- ⁇ interferon-gamma
  • TNF- alpha TNF- ⁇
  • NO - Nitric oxide
  • viability viability
  • proliferative changes in the immune cells and the production were evaluated by analyzing.
  • 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.
  • mice 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 °C, 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 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.
  • 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 ⁇ l 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.
  • 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.
  • cytokines TNF- ⁇ , IFN- ⁇ , IL-12
  • 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).
  • HRP horseradish peroxidase
  • 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 ⁇ l 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).
  • 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 ⁇ l 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).
  • N (%) is the cell growth ratio
  • C p is the absorbance after killing
  • C s is the absorbance after no treatment.
  • MTT assay Metal thiazolinlinyldiphenyl tetrazolium bromide assay
  • 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 ⁇ g / ml) for 24 hours.
  • MTT 3- [4,5-dimethyl-thiazol] -2,5-diphenyl-tetrazolium bromide
  • solvent DPBS Dulbecco's Phosphate-Buffered Saline, 1X
  • DMSO dimethyl sulfoxide
  • PBS phosphate buffered saline
  • the oil red o dye was added 500 ⁇ l 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 ⁇ l 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 ⁇ m filter.
  • Triglycerides in fat cells Tri - glycerides A
  • 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 °C. 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 °C cryogenic freezer.
  • PBS phosphate buffered saline
  • cryoprotectant 25% glycerin + 10% skim milk powder
  • 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.
  • the vial was taken out of the cryogenic freezer and dissolved, followed by seed culture of lactic acid bacteria.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ m diameter and a 6 mm channel radius, an Al-Al ceramic hollow fiber membrane filter) and an 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 ⁇ m diameter and a 6 mm channel radius) before being added to the incubator.
  • 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 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.
  • the volume of the culture solution is reduced at a constant rate while passing through the first membrane filter.
  • group 1 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.
  • the lactic acid bacteria live bacteria obtained in Example 1 was circulated three times at 121 °C 3 seconds using an ultra-high temperature continuous sterilizer, to prepare lactic acid bacteria bacteria (Example 2-1).
  • the lactic acid bacteria live bacteria obtained in Example 1 was circulated for 5 minutes at 110 °C using an ultra-high temperature continuous sterilizer, to prepare lactic acid bacteria bacteria (Example 2-2, Figure 5b).
  • the lactic acid bacteria live bacteria obtained in Example 1 was circulated for 3 minutes at 65 °C, 85 °C and 120 °C using an ultra high temperature continuous sterilizer, to prepare lactic acid bacteria bacteria (Example 2-3, 7).
  • 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).
  • the second membrane filter (0.01 ⁇ m to 0.1 ⁇ m 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.
  • lactic acid bacteria are aggregated in the process of killing the lactic acid bacteria to form a lump of lactic acid bacteria.
  • the size of the lactic acid bacteria killing bacteria formed immediately after the killing of lactic acid bacteria is 20 ⁇ m To 200 ⁇ m (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 ⁇ m to 3.0 ⁇ m or less (Fig. 9).
  • the size distribution of the washed, dried and powdered lactic acid bacteria as described above is shown in FIG. 9.
  • the size of the powdered dead bacteria showed a size distribution of 0.5 ⁇ m to 3.0 ⁇ m, and furthermore, the size of 60% to 100% of the powdered dead bacteria appeared to be 1.0 ⁇ m or less. .
  • 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.
  • the stirring speed was maintained at 30 rpm and the temperature at 32 ° C.
  • 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.
  • the live bacteria of the lactic acid bacteria were sampled at regular intervals and analyzed.
  • the live bacteria of the lactic acid bacteria were heat-treated at 110 ° C. for 5 minutes (Comparative Example 1-1, FIG. 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.
  • the live bacteria of the lactic acid bacteria were circulated by circulating three times for 3 seconds at 120 °C (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.
  • lactic acid bacteria were dried and powdered in the same manner as in Example 4.
  • 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 is represented by the following equation 4.
  • 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.
  • 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.
  • Q h was the amount of medium (L) consumed per hour and V is the total volume (L) of the bioreactor.
  • 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.
  • 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).
  • the lactic acid bacteria live bacteria 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.
  • 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 ⁇ m, while the size of the lactic acid bacteria live bacteria according to Example 1 has a smaller size of up to 4.0 ⁇ m Distribution.
  • the size was reduced to 3.0 ⁇ m, which indicates that the value is significantly smaller than 4.0 ⁇ m of Comparative Example 1.
  • 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%.
  • 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.
  • 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- ⁇ , INF- ⁇ , IL-12) in the mouse blood was measured. An in-vivo test was performed to show in FIG. 12.
  • 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- ⁇ , INF by - ⁇ , 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 ⁇ g / 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 ⁇ g / 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.
  • 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.
  • 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.
  • 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 ⁇ g / 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 ⁇ g / ml) of the lactic acid bacteria bacterium powder ( 15A).
  • Experimental Example 7 shows that LM1004 has an effect of inhibiting lipid accumulation in adipocytes.

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Abstract

La présente demande concerne un procédé de contrôle de la taille des lactobacilles comprenant le passage d'une solution de culture de lactobacilles à travers un filtre à membrane.
PCT/KR2017/012087 2016-12-02 2017-10-30 Procédé de contrôle de la taille des lactobacilles à l'aide d'un filtre à membrane Ceased WO2018101612A1 (fr)

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KR20100110345A (ko) * 2008-02-04 2010-10-12 도레이 카부시키가이샤 연속 발효에 의한 유산의 제조방법
KR20100088894A (ko) * 2009-02-02 2010-08-11 에이엠바이오 (주) 멤브레인 생물반응기를 이용한 고농도 유산균의 생산방법 및 유산균 동결건조 분말의 제조방법
KR20120108198A (ko) * 2011-03-23 2012-10-05 충북대학교 산학협력단 내부필터시스템이 구비된 생물반응기를 이용한 유산균의 고농도 배양 및 대사산물의 생산 방법
KR20120135450A (ko) * 2011-06-03 2012-12-14 정일선 세라믹 막 분리 배양기
KR101794635B1 (ko) * 2016-11-30 2017-11-07 주식회사 락토메이슨 막 필터를 이용한 고농도 사균의 제조방법 및 이의 제조방법으로 제조된 사균

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WO2024252017A1 (fr) * 2023-06-08 2024-12-12 Dsm Ip Assets B.V. Nouveau procédé de production de lactobacillus rhamnosus

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