JP2012229258A - Nano-type lactic acid bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a lactic acid bacterium having a favorable particle diameter for Th1 induction, excellent in the productivity of INF-α, and excellent in dispersibility in water.SOLUTION: A bacterial cell of a nano-type lactic acid bacterium is obtained by adjusting a pH in a medium in a culturing step and a processing step of the lactic acid bacterium to a neutral range, wherein a mode value in a particle size distribution is 1.0 μm or less and the lactic acid bacterium is lactobacillus brevis.

Description

  The present invention relates to a nano-type lactic acid bacterium having an action of enhancing the ability to produce interferon α from antigen-presenting cells.

In recent studies, it is known that naive T cells (hereinafter abbreviated as Th0) functionally differentiate into type I T cells (hereinafter abbreviated as Th1) and type II T cells (hereinafter abbreviated as Th2). ing.
The immune response by Th1 cells induces cellular immunity, and phagocytosis of mononuclear cells such as macrophages and lymphocytes occurs. On the other hand, the immune response by Th2 cells induces humoral immunity and sterilization with antibodies occurs. Th1-type cytokines suppress Th2, and conversely, Th2-type cytokines suppress Th1, and the two are related to each other in order to maintain the overall immunity balance.

Interferon α (IFN-α) is a dendritic cell (antigen-presenting cell) during infection by viruses, intracellular parasitic bacteria such as Mycobacterium tuberculosis, Salmonella, Listeria, and leprosy, and intracellular parasitic fungi such as cryptococcus. It is a cytokine secreted from.
Interleukin 12 (IL-12) is a cytokine secreted from antigen-presenting cells such as dendritic cells and macrophages. Natural killer cells (NK cells) that directly attack cancer cells and rack cells (LAK cells). ), Which is known as a very powerful immunologically active substance that activates killer T cells (CTL cells) and enhances production of IFN-γ.

  Both of these IFN-α and IL-12 are cytokines that induce Th1 cells, but the receptor (TRL: Toll-like receptor) expressed in antigen-presenting cells (in the case of IL-12: TLR1) , TLR3, TLR5, TLR9; IFN-α: TLR7, TLR9).

  By the way, as for lactic acid bacteria that enhance IFN-α production from antigen-presenting cells or components thereof, Lactobacillus brevis powder (see Patent Document 1: JP-A-6-206826), lactic acid bacteria belonging to the genus Enterococcus or the same Processed products (see Patent Document 2: JP-A-8-259450), constituent extracts of Lactobacillus brevis strain FERM BP-4693 (Patent Document 3: see JP-A-9-188627) and the like have been reported. .

Normally, it is natural to select a strain having a high IFN-α-producing ability, and it is not necessary to intentionally change a strain having a low activity to a strain having a high activity.
However, for businesses that have provided Lactobacillus brevis bacteria on the market for many years, it is an urgent desire to enhance the ability of lactic acid bacteria to produce IFN-α.

Until now, lactic acid bacteria are usually taken in the form of fermented milk or yogurt. Antigen taken from the mouth is taken up by M cell phagocytosis on Peyer's patches.
Therefore, many studies have been conducted on the relationship between the size (particle diameter) of particles and the incorporation into Peyer's plate from the interest of how large it can be taken from Peyer's plate.

As a result, when the particle size exceeds 10 μm, phagocytosis by M cells is remarkably reduced (see Non-Patent Document 1), and the maximum diameter of particles passing through Peyer's patch is 10 μm when the particle material is polylactide ( It is clear that 15 μm (see Non-Patent Document 3) in the case of polystyrene, and 21 μm (see Non-Patent Document 4) in the case of biodegradable polylactic acid.
From these results, it can be seen that the size of particles that can pass through the Peyer plate is at most about 20 μm.

In addition, according to an experiment in which biodegradable polylactic acid beads sensitized with an antigen were orally administered to rats, the particle diameter at which the rate at which an antigen-specific antibody was produced by Th2 induction was highest was 4 μm for IgG, IgA In this case, the particle diameter is 7 μm, and the preferable particle diameter for Th2 induction is about 3 to 7 μm (see Non-Patent Document 4).
However, the preferred particle size for Th1 induction has not been clarified so far.

JP-A-6-206826 JP-A-8-259450 JP-A-9-188627

Tabata Y, Ikada Y. Adv Polym Sci 94: 107-141, 1990. Eldridge JH. Et al. J Controlled Rel 11: 205-214, 1990. Eldridge JH. Et al. Molec Immun 28: 187-194, 1991. Tabata Y. et al. Vaccine 14: 1677-1685, 1996.

  The present invention has been made in view of such circumstances, and provides a lactic acid bacterium having a particle size preferable for Th1 induction, excellent INF-α production ability, and excellent dispersibility in water. With the goal.

The present inventors isolated lactic acid bacteria related to longevity from Kyoto's pickles, Nagano's pickles, Georgian mariami and Mazzoni-like, Mongolian horse milk and various fermented milk products. The relationship between the size of lactic acid bacteria and the ability to produce IL-12 and IFN-α from antigen-presenting cells was analyzed.
As a result, as shown in FIG. 1A, the ability to produce IL-12 from antigen-presenting cells by stimulation with lactic acid bacteria increased when the size of lactic acid bacteria was reduced to 1 μm or less (see Reference Example 1).
On the other hand, also in the case of IFN-α production ability, as shown in FIG. 1B, it increased as the size of lactic acid bacteria decreased to 1 μm or less (see Reference Example 1).

However, some strains were negatively correlated with IL-12 and IFN-α production ability (see FIG. 2). For example, strains such as EF, KH1, and KH3 have high IL-12 production ability and low IFN-α production ability. Conversely, strains such as LL12 and ML4 have low IL-12 production ability and high IFN-α production ability.
On the other hand, SNK was characterized by relatively high ability to produce IL-12 and IFN-α.
This is a finding that cannot be conceived from the prior art (Japanese Patent Application No. 2007-30324), and is presumably due to a difference in recognition of lactic acid bacteria by receptors expressed in antigen-presenting cells.

  As a result, in Lactobacillus having a particle diameter of nearly 9 μm, such as Lactobacillus brevis strain FERM BP-4693 (indicated by LBR in Table 1 and FIG. 1-2), it is compared with that of about 1 μm. It was revealed that the ability to produce IFN-α was extremely inferior.

Therefore, in order to solve the above-mentioned problems, the present inventor diligently studied conditions for reducing the mode value in the particle size distribution of lactic acid bacteria and preventing reaggregation of the bacteria.
In that process, paying attention to the fact that the surface of lactic acid bacteria is positively charged, and adjusting the pH in the culture process and processing process to the neutral range, the mode value in the particle size distribution of lactic acid bacteria (below) It was found that the particle size is sometimes reduced to 1.0 μm or less.
In general, it is known that the form of lactic acid bacteria changes depending on various conditions during cultivation, but the knowledge that the size can be adjusted to 1.0 μm or less by controlling the pH in the culturing and processing steps. Has not been known to those skilled in the art.

The present inventor has also found that the ability to produce IFN-α from antigen-presenting cells can be remarkably enhanced with the cells.
As described above, the present inventor adjusted the particle size of lactic acid bacteria to 1.0 μm or less through culturing and processing at a neutral pH even for lactic acid bacteria having a normal particle size larger than 1.0 μm. And the present inventors have found that this lactic acid bacterium can enhance the ability to produce IFN-α from antigen-presenting cells and improve the immunostimulatory effect.

Recently, dry powder enriched with dead lactic acid bacteria has been found on the market. This is intended to enable intake of a large amount of lactic acid bacteria in a small amount.
However, even if the original particle size of lactic acid bacteria in such a dry powder is 1.0 μm or less, if the powder is simply dried concentrated bacteria, the particles will adsorb when placed in water. -Aggregates to form a lump (refer to Reference Example 2).
In the first place, under normal culture conditions, lactic acid bacteria having a particle size of 1.0 μm or less are unlikely to exist (see Table 1).

That is, the present invention provides the following nano-type lactic acid bacteria.
1. A cell of nano-type lactic acid bacteria obtained by adjusting the pH of the medium in the culture process and processing process of lactic acid bacteria to a neutral range, and the mode value in the particle size distribution is 1.0 μm or less,
A microbial cell of a nano-type lactic acid bacterium, wherein the lactic acid bacterium is Lactobacillus brevis.
2. A cell of 1 nano-type lactic acid bacterium obtained by adjusting the pH of the medium to 5-8.
3. A cell of 1 or 2 nano-type lactic acid bacteria obtained by using the culture medium in the culturing step as a source of energy and glucose as an energy source, and obtaining the time when the glucose is consumed as a culture end point.
4). In the processing step, a nano-type lactic acid bacterium cell body obtained by adding a dispersant or an excipient to a lactic acid bacterium, followed by freeze-drying or spray-drying.
5. The composition which has the immunostimulatory effect containing the microbial cell of nano type lactic acid bacteria in any one of 1-4.
6). Food / beverage, feed, cosmetics or pharmaceuticals containing any one of 1 to 4 nano-type lactic acid bacteria or 5 compositions.
7). The interferon alpha production ability enhancer containing the microbial cell of any one of 1-4 nano type lactic acid bacteria.
In addition, the present invention relates to the following nano-type lactic acid bacteria.
[1] A nano-type lactic acid bacterium having a mode value in a particle size distribution of 1.0 μm or less.
[2] The cell of the nano-type lactic acid bacterium according to [1], which is obtained by adjusting the pH of the medium in the lactic acid bacterium culture process and processing step to a neutral range.
[3] The nano-type lactic acid bacteria of [2] obtained by adjusting the pH of the medium to 5 to 8.
[4] Nano-type lactic acid bacteria according to [2] or [3], wherein glucose is added to the medium in the lactic acid bacteria culture step.
[5] The nano-type of [1] obtained by adding a dispersing agent or an excipient to a culture solution containing a medium having a pH adjusted to a neutral range and a bacterium and dispersing the dispersion, followed by freeze drying or spray drying Lactic acid bacteria.
[6] The cell of the nano-type lactic acid bacterium according to any one of [1] to [5], which is a Lactobacillus lactic acid bacterium.
[7] The lactic acid bacterium according to [6], wherein the Lactobacillus lactic acid bacterium is Lactobacillus brevis.
[8] The nano-type lactic acid bacterium of [7], wherein the Lactobacillus brevis strain is FERM BP-4693.
[9] A composition having an immunostimulatory effect, comprising the cells of the nano-type lactic acid bacteria according to any one of [1] to [8].
[10] A food / beverage, feed, cosmetic or pharmaceutical product containing the lactic acid bacterium of any one of [1] to [8] or the composition of [9].
[11] The method for producing a cell of a nano-type lactic acid bacterium according to [1], wherein the pH of the medium in the lactic acid bacteria culture and processing steps is adjusted to a neutral range.
[12] The method for producing cells of nano-type lactic acid bacteria according to [11], wherein the pH of the medium is adjusted to 5 to 8.
[13] A nano-type lactic acid bacterium according to [11], which is dispersed by adding a dispersant or an excipient to a culture solution containing a medium having a pH adjusted to a neutral range and a bacterium, and then lyophilized or spray-dried. Body manufacturing method.

According to the present invention, the particle size of lactic acid bacteria can be adjusted to 1.0 μm or less through culturing and processing at a neutral pH even for lactic acid bacteria having a normal particle size larger than 1 μm.
The cells of the nano-type lactic acid bacterium thus obtained can be said to be very useful cells because they can enhance the ability to produce IFN-α from antigen-presenting cells and improve the immunostimulatory action.

It is the figure which showed the relationship between the microbial cell particle size and IL-12 and IFN- (alpha) production ability. It is the figure which showed the correlation of IL-12 and IFN- (alpha) production ability by lactic acid bacteria stimulation. It is the figure which compared the microbial cell particle size. It is the figure which showed the particle size distribution (A: frequency%, B: integration%) of lactic acid bacteria powder. It is the figure which showed the microbial cell particle size and IFN- (alpha) production ability of nano type | mold lactic acid bacteria Labre.

Hereinafter, the present invention will be described in more detail.
The cells of nano-type lactic acid bacteria according to the present invention have a mode value (particle size) of 1.0 μm or less in the particle size distribution.
In the present invention, the "mode value in the particle size distribution" is a value serving as an index representing the size of the bacterium, and the particle diameter that maximizes the relative frequency in the particle size distribution when the particle size of the bacterial cell is measured. Say.

  Specific examples of the lactic acid bacterium used as a raw material for the “bacteria of the nano-type lactic acid bacterium” of the present invention include Lactobacillus acidophilus, L. gasseri, and Lactobacillus mari (L. mari). Lactobacillus plantarum (L. plantarum), Lactobacillus buchneri (L. buchneri), Lactobacillus casei (L. casei), Lactobacillus johnsonii (L. johnsonii), Lactobacillus gallinarum (L. gallinarum), Lactobacillus amylovorus (L. amylovorus), Lactobacillus brevis (L. brevis), Lactobacillus rhamnosus (L. rhamnosus), Lactobacillus Lactobacillus bacteria such as Kefir, L. paracasei, L. crisptus, Streptococcus thermophilus, Streptococcus such as Streptococcus thermophilus Lactococcus bacteria such as Lactococcus lactis, Enterococcus faecalis, Enterococcus faecium, etc., Bifidobacteria Bifidobacterium・ Longham (B. longum), bifi B. adolescentens, B. infantis, B. breve, B. catenatum, B. catenatum, etc. Examples include bacteria belonging to the genus Fidobacterium.

  The form of the cells of the nano-type lactic acid bacteria of the present invention may be live or dead, but in the case of live bacteria, there is a possibility of causing a change in form at the time of delivery or display after product manufacture. A killed bacteria that does not cause odor is preferred.

Lactic acid bacteria are known to change in form due to stress when the growth environment during culture becomes poor.
Therefore, in the present invention, by controlling the culture and processing conditions, the lactic acid bacteria are grown while maintaining the form of the lactic acid bacteria to be constant, and the nano-type lactic acid bacteria having the mode value in the particle size distribution described above are manufactured.
Specifically, as mentioned above, the surface of lactic acid bacteria is positively charged (positive, positive), and the membrane is stabilized by adjusting the pH in the culture and processing steps to a neutral range. By doing so, it prevents the double bacteria state in which the dividing bacteria are joined and the re-adsorption of the bacteria.

Here, as a method of “adjusting pH to a neutral range”, neutralization with an acid such as lactic acid or an alkali such as sodium hydroxide can be mentioned.
In the present invention, “adjusting the pH of the medium in the culturing step and the processing step to the neutral range” not only adjusts the pH of the culturing step to the neutral range, but also sterilizes the cells after completion of the culture. It means that the pH in the process (processing step) such as washing and concentration is also adjusted to the neutral range.
5-8 are preferable and, as for pH in a culture process and a processing process, 5.5-7.5 are more preferable.

Moreover, as an energy source in the nutrient composition of a culture medium, glucose with the highest energy utilization is preferable, and it is preferable that the addition amount shall be about 5-10 mass% in a culture medium.
In addition, the change of a fungal form by the stress which comes from nutrient depletion can be prevented by setting the time of glucose consumption as the culture end point.

Furthermore, it is preferable that the “bacteria of nano-type lactic acid bacteria” of the present invention have been subjected to a dispersion treatment.
The method for the dispersion treatment is not particularly limited, and examples thereof include a method of dispersing a bacterial culture solution with a high-pressure homogenizer of about 150 kgf / cm ² (1.5 MPa) in a wet manner.
In this case, it is preferable to add a known dispersing agent or excipient in advance to the culture solution, whereby the bacterial cells can be effectively prevented from reaggregating.
Although the addition amount of the dispersing agent and excipient | filler to be used changes with the characteristics of a microbial cell, 1-100 times amount is preferable with respect to a microbial cell in conversion of mass, and 2-20 times amount is more preferable.
Suitable dispersing agents and excipients include trehalose, dextrin, skim milk and the like.

  In the case of finally obtaining the “nano-type lactic acid bacteria” of the present invention as a powder, the cells are treated with a known dispersant / excipient so that the cells do not reaggregate, and then lyophilized. Or spray drying. Thereby, the microbial cell powder excellent in the dispersibility to water can be obtained.

The cells of the nano-type lactic acid bacteria of the present invention described above have been refined to a nanometer (nm) size with a particle size of 1.0 μm or less.
Moreover, this microbial cell is made into a dry powder by the said method, and the microbial cell particle size at the time of resuspending the said powder in a physiological digestive liquid also maintains 1.0 micrometer or less. The physiological digestive fluid means artificial gastric juice or intestinal fluid prepared by a known method.

The cells of the nano-type lactic acid bacterium of the present invention can be used as it is, but in general, various ingredients are added and blended in order to increase the flavor, make it a required shape, etc. The final product is added.
Examples of the components to be added and mixed include various sugars, emulsifiers, sweeteners, acidulants, fruit juices and the like.

  More specifically, sugars such as glucose, sucrose, fructose, and honey; sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, and palatinit; emulsifiers such as sucrose fatty acid ester, glycerin sugar fatty acid ester, and lecithin . In addition, various types of vitamins such as vitamin A, vitamin B, vitamin C and vitamin E, herbal extracts, cereal ingredients, vegetable ingredients, milk ingredients, etc. can be blended to obtain an excellent flavor Th1 inducer. Can do.

  In addition, flavors include yogurt, berry, orange, quince, perilla, citrus, apple, mint, grape, pair, custard cream, peach, melon, banana, tropical, herbal, tea And coffee-based flavors, which can be used alone or in combination of two or more. Although the addition amount of a flavor is not specifically limited, 0.05-0.5 mass% in a microbial cell from a flavor surface, Especially about 0.1-0.3 mass% is preferable.

  The nano-type lactic acid bacteria of the present invention described above can be made into a product in any form such as solid or liquid. Specifically, various pharmaceutically acceptable salts, excipients, preservatives, coloring agents, flavoring agents, and the like, as well as various beverages, granules, tablets, capsules, and the like, by known methods in the pharmaceutical or food manufacturing field. It can be commercialized as a form.

  Moreover, the microbial cell of the nano type | mold lactic acid bacteria of this invention can be utilized for health food. The health food means a food that is more active than normal food and intended for health, health maintenance and promotion. The form may be any of liquid, semi-solid, and solid, and specific examples thereof include confectionery such as cookies, rice crackers, jelly, yokan, yogurt, and manju; soft drinks, nutritional drinks, soups and the like.

  Furthermore, the cells of the nano-type lactic acid bacteria of the present invention include lotions (skin lotions), cosmetic creams, emulsions, lotions, packs, skin milk (emulsions), gels, powders, lip balms, lipsticks, undermakes. Up, foundation, sun care, bath preparation, body shampoo, body rinse, soap, cleansing foam, ointment, patch, jelly, aerosol, etc., can be used as a skin external preparation.

In addition, various components and additives usually used in cosmetics, quasi-drugs, and pharmaceuticals as exemplified below can be appropriately blended into the cells of the nano-type lactic acid bacteria of the present invention as necessary. .
That is, humectants such as glycerin, petrolatum, urea, hyaluronic acid, heparin; PABA derivatives (paraaminobenzoic acid, Escalol 507 (Asp Japan)), cinnamic acid derivatives (neoheliopan, pulsol MCX (DSM nutrition) Japan Co., Ltd.), Sungard B (Shiseido Co., Ltd.), salicylic acid derivatives (octyl salicylate, etc.), benzophenone derivatives (ASL-24, ASL-24S (Shonan Chemical Service), etc.), dibenzoyl Methane derivatives (Pulsol A, Pulsol DAM (DSM Nutrition Japan Co., Ltd., etc.), heterocyclic derivatives (Tinuvin, etc.), UV absorbers / scattering agents such as titanium oxide; disodium edetate, trisodium edetate, citric acid , Sodium citrate, tartaric acid, Metal sequestering agents such as sodium tartrate, lactic acid, malic acid, sodium polyphosphate, sodium metaphosphate, gluconic acid; sebum inhibitors such as salicylic acid, sulfur, caffeine, tannin; benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate Anti-inflammatory agents such as diphenhydramine hydrochloride, tranexamic acid, guaiazulene, azulene, allantoin, hinokitiol, glycyrrhizic acid and salts thereof, glycyrrhizic acid derivatives, glycyrrhetinic acid; vitamin A, vitamin B group (B1, B2, B6, B12, B15), folic acid, nicotinic acids, pantothenic acids, biotin, vitamin C, vitamin D group (D2, D3), vitamin E, ubiquinones, vitamins such as vitamin K (K1, K2, K3, K4) Aspartic acid, Amino acids and their derivatives such as glutamic acid, alanine, lysine, glycine, glutamine, serine, cysteine, cystine, tyrosine, proline, arginine, pyrrolidone carboxylic acid; retinol, tocopherol acetate, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, kouji Whitening agents such as acid, ellagic acid and placenta extract; antioxidants such as butylhydroxytoluene, butylhydroxyanisole and propyl gallate; astringents such as zinc chloride, zinc sulfate, zinc carbonate, zinc oxide and aluminum potassium sulfate; Glucose, fructose, maltose, sucrose, trehalose, erythritol, mannitol, xylitol, lactitol, and other sugars; licorice, chamomile, maronier, yukinoshita, glaze, karin, ougon, owe Click, Coptis, herba houttuyniae, other various kinds plant extracts such as ginkgo biloba, oil components, surfactants, thickeners, alcohols, powder components, can be as appropriate blended dye.

  Hereinafter, although a reference example and an example are given and the present invention is explained more concretely, the present invention is not limited to the following examples. The particle size of lactic acid bacteria was measured with a particle size distribution measuring device (Shimadzu Corporation, SALD-3100). IL-12 and IFN-α produced from macrophages by stimulation with lactic acid bacteria were measured using a commercially available ELISA kit.

[Reference Example 1]
[Preparation of dead cells]
As shown in Table 1, lactic acid bacteria related to longevity are isolated from Kyoto's pickles, Nagano's pickles, Georgian mariami and Mazzoni-like, Mongolian horse milk and various fermented milk products. Then, the cells were cultured at 36.5 ° C. for 48 hours without adjusting the pH during the culture using MRS medium. After completion of the culture, the culture solution was sterilized by heating at 80 ° C. for 10 minutes, and the cells were washed with PBS to prepare a cell concentration of 10 mg / ml.
In addition, LBR in the table refers to Lactobacillus brevis strain FERM BP-4693. The Lactobacillus brevis strain FERM BP-4693 can be obtained from the Independent Administrative Institution Product Evaluation Technology Base Organization Patent Microorganism Depositary Center.

[Relationship between cell particle size and ability to produce IL-12 and IFN-α]
The relationship between the cell size of lactic acid bacteria and the ability to produce IL-12 and IFN-α is shown in FIGS. 1A and 1B, respectively.
It has been clarified that the production ability of both IL-12 and IFN-α cytokines is remarkably enhanced when the size (particle diameter) of lactic acid bacteria is reduced to about 1 μm (Table 1 and FIG. 1). reference).
However, as shown in FIG. 2, strains having a negative correlation were observed in the production ability with IL-12 and IFN-α. For example, strains such as EF, KH1, and KH3 have high IL-12 production ability and IFN. -Α production ability is low, conversely, strains such as LL12 and ML4 have low IL-12 production ability and high IFN-α production ability, whereas SNK has relatively high production ability of IL-12 and IFN-α. The following features were recognized.

[Example 1]
[Preparation of nano-type lactic acid bacteria EF]
Lactic acid bacteria Enterococcus faecalis strain EF was cultured at 36.5 ° C. in a known nutrient medium supplemented with 5% by weight glucose and adjusted to pH 6.5 at the time of cultivation with 20% by weight sodium hydroxide aqueous solution. The time point of consumption was defined as a culture end point (culture process).
After completion of the culture, the culture solution was sterilized by heating at 80 ° C. for 10 minutes, and then the cells were washed with PBS and adjusted to a cell concentration of 10 mg / ml (processing step). The pH during the processing step was maintained at 6.5.
[Comparison of cell size]
The cell size of the lactic acid bacteria EF prepared in Reference Example 1 and Example 1 was measured. The result is shown in FIG.
As shown in FIG. 3, the cell size by non-neutralization culture was 1.215 and larger than 1 μm, whereas the cell size by neutralization culture was 0.701 and 1.0 μm or less. I understand.

[Reference Example 2]
[Preparation of dry powder of dead bacteria]
Lactic acid bacteria Enterococcus faecalis strain EF having an original size of lactic acid bacteria of 0.6 μm (FIG. 4A) and an integrated distribution of particles of 2 μm or less capable of selectively inducing cytokines (99% (FIG. 4B)) is used as the method of Example 1 The cells were concentrated from the culture solution and spray-dried without adding excipients to obtain powder.
[Comparison of cell size]
The powder prepared above was dispersed again in water. As a result, as shown in FIG. 4A, the particle size distribution showed a gentle particle size distribution up to 100 μm. The breakdown is as shown in the cumulative distribution of FIG. 4B. About 50% of particles up to 20 μm that pass through Peyer's patches, about 14% of particles of 3-7 μm that induce Th2 cells, and further induce Th1 cells. Particles of 2 μm or less were at most 1%. In this case, particles that induce both Th1 / Th2 responses are mixed, which is not a physiologically preferable situation.

In addition, assuming an in vivo digestive action, even when this is treated with an artificial gastric fluid or intestinal fluid prepared by a known method, the cumulative distribution of particles of 2 μm or less that induce Th1 cells increases only to 10% at most. (FIG. 4B).
There is a concern that this may lead to a result that the original function of lactic acid bacteria is not fully exhibited (about 10% at most).

[Example 2]
[Preparation of nano-type lactic acid bacteria Lactobacillus brevis]
Lactic acid bacterium Lactobacillus brevis strain FERM BP-4693 is cultured at 36.5 ° C. in a known nutrient medium supplemented with 5% by weight glucose and adjusted to pH 6.5 with 20% by weight sodium hydroxide aqueous solution. Then, the time point when the glucose consumption was completed was defined as the culture end point (culture step).
After completion of the culture, the culture solution is sterilized by heating at 80 ° C. for 10 minutes, the cells are washed with PBS, 4 times the amount of dextrin is added as an excipient to the cells and dispersed with a mixer. Then, the sample was freeze-dried to prepare a sample, which was again suspended in PBS so that the bacterial cell concentration was 10 mg / ml (processing step). The pH during the processing step was maintained at 6.5.

[Comparison of cell size and IFN-α production ability]
The cell size and IFN-α production ability of Lactobacillus brevis strain FERM BP-4693 prepared in Reference Example 1 and Example 2 were measured. The result is shown in FIG.
As shown in FIG. 5, in the case of non-neutralization culture (labeled LBR), the cell size was 8.8 μm and the IFN-α production ability was 16.8 pg / ml, whereas neutralization culture was used. (Indicated as NANO-LBR), the cell size is 0.7 μm and 1.0 μm or less, and the IFN-α production ability is 92.9 pg / ml, which is 5. It can be seen that it is enhanced 5 times.

  From the above results, by adjusting the pH in the culturing and processing steps to a neutral range, the cell particle size can be refined to 1.0 μm or less, and further about 4 times the amount of excipient in terms of mass in the cell. It was confirmed that a cell powder excellent in dispersibility can be obtained by adding a dispersion treatment after lyophilization and freeze-drying, and that this powder can efficiently produce interferon α from macrophages.

Claims (7)

A cell of nano-type lactic acid bacteria obtained by adjusting the pH of the medium in the culture process and processing process of lactic acid bacteria to a neutral range, and the mode value in the particle size distribution is 1.0 μm or less,
A microbial cell of a nano-type lactic acid bacterium, wherein the lactic acid bacterium is Lactobacillus brevis.   The microbial cell of the nano type | mold lactic acid bacteria of Claim 1 obtained by adjusting the pH of the said culture medium to 5-8.   The microbial cell of nano-type lactic acid bacteria according to claim 1 or 2, wherein the culture medium in the culturing step contains glucose as an energy source, and is obtained at the time when the glucose is consumed as a culture end point.   The microbial cell of the nano type | mold lactic acid bacteria of Claim 1 obtained by adding a dispersing agent or an excipient | filler to lactic acid bacteria in the said process process, and then freeze-drying or spray-drying.   The composition which has the immunostimulatory effect containing the microbial cell of nano type | mold lactic acid bacteria of any one of Claims 1-4.   Food / beverage, feed, cosmetics or a pharmaceutical containing the nano-type lactic acid bacteria according to any one of claims 1 to 4 or the composition according to claim 5.   An interferon α-producing ability enhancer containing the nano-type lactic acid bacteria according to any one of claims 1 to 4.

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