Lactic acid bacteria cell wall disrupted product and method for producing lactic acid bacteria cell wall disrupted product

Abstract

Provided is a lactic-acid-bacteria-derived material that is suitable as a compounding material, etc., for foods, pharmaceuticals, cosmetics, etc. A disrupted product of lactic acid bacteria, wherein the lactic acid bacteria cell wall disrupted product is obtained by removing contents other than the cell wall from the lactic acid bacteria. Also, a method for producing a lactic acid bacteria cell wall disrupted product, the method involving disrupting lactic acid bacteria using a wet atomization means and performing solid-liquid separation to recover the solid phase fraction. This lactic acid bacteria cell wall disrupted product can be suitably utilized as a compounding material, etc., in cosmetics.

Description

Technical Field

[0001] The present invention relates to a crushed lactic acid bacteria cell wall useful as a compounding material for foods, pharmaceuticals, cosmetics, etc.

Background Art

[0002] Conventionally, it has been clarified that various lactic acid bacteria are useful as compounding materials for foods, pharmaceuticals, cosmetics, etc.

[0003] That is, for example, Patent Document 1 discloses a leukopenia preventive and therapeutic agent containing, as an active ingredient, a microbial cell or a processed product thereof belonging to the genus Enterococcus.

[0004] Further, Patent Document 2 discloses a cytotoxicity reducing agent for anticancer agents containing, as an active ingredient, a microbial cell or a processed product thereof belonging to the genus Enterococcus.

[0005] Further, Patent Document 3 discloses a prophylactic, ameliorating or therapeutic agent for oral diseases containing, as an active ingredient, a microbial cell or a microbial cell culture or an extract thereof of one or more lactic acid bacteria selected from Lactobacillus rhamnosus KO3 strain, Lactobacillus casei YU3 strain and Lactobacillus paracasei YU4 strain.

[0006] Further, Patent Document 4 discloses a biological antioxidant ability activator containing a microbial cell of a lactic acid bacterium belonging to the genus Enterococcus.

[0007] Further, Patent Document 5 discloses a circadian rhythm improving agent containing, as an active ingredient, a microbial cell of Lactobacillus brevis or a processed product thereof.

[0008] Further, Patent Document 6 discloses a cancer metastasis inhibitor containing at least one selected from the group consisting of a microbial cell of a lactic acid bacterium belonging to the genus Enterococcus and a microbial cell component thereof.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0010] However, many of the existing materials using lactic acid bacteria cells are mixtures of cell wall components and cytoplasmic components, and it is difficult to completely separate the two. Therefore, due to the decomposition or denaturation of unstable cytoplasmic components, etc., there has been a problem that the quality of the formulated products is significantly affected. On the other hand, a method of purifying only the cell wall components by enzyme treatment has also been reported, but there are problems such as the applicable range being limited due to differences in the enzyme sensitivity of lactic acid bacteria. Therefore, an object of the present invention is to provide a lactic acid bacteria-derived material improved against such problems.

Means for Solving the Problems

[0011] As a result of intensive research to achieve the above object, the present inventors have found that by uniformly refining lactic acid bacteria to have a certain particle size, the contents such as proteins can be sufficiently removed, and thus the present invention has been completed.

[0012] That is, the present invention firstly provides a crushed product of lactic acid bacteria, which is characterized in that the content other than the cell wall of the lactic acid bacteria has been removed, namely, a crushed product of lactic acid bacteria cell wall.

[0013] In the above-mentioned crushed product of lactic acid bacteria cell wall, it preferably has a particle size with a median diameter of 0.1 μm or more and 0.5 μm or less.

[0014] In the above-mentioned crushed product of lactic acid bacteria cell wall, the content of one or more amino acids among the types of amino acids that do not constitute peptidoglycan present in the cell wall of the lactic acid bacteria is preferably reduced to 1 / 3 or less of the content of the amino acid contained in the raw material lactic acid bacteria in terms of mass molar concentration unit (mol / g).

[0015] In the above-mentioned crushed product of lactic acid bacteria cell wall, the content of one or more amino acids selected from the group consisting of proline, arginine, threonine, and phenylalanine is preferably reduced to 1 / 3 or less of the content of the amino acid contained in the raw material lactic acid bacteria in terms of mass molar concentration unit (mol / g).

[0016] Secondly, the present invention provides a method for producing a crushed product of lactic acid bacteria cell wall, which is characterized by crushing lactic acid bacteria by wet atomization means, performing solid-liquid separation, and recovering the solid phase fraction.

[0017] In the method for producing the above-mentioned crushed product of lactic acid bacteria cell wall, the lactic acid bacteria to be crushed by the wet atomization means are preferably prepared as a sample in a solution containing a buffer and / or a surfactant.

[0018] In the method for producing the above-mentioned crushed product of lactic acid bacteria cell wall, the crushing by the wet atomization means is preferably repeated a plurality of times by the means, and then the solid-liquid separation treatment is performed.

[0019] In the method for producing the disrupted lactic acid bacteria cell wall, the pressure condition for disruption by the wet micronization means is preferably 100 MPa or more and 250 MPa or less.

Advantages of the Invention

[0020] According to the disrupted lactic acid bacteria cell wall of the present invention, proteins and the like derived from lactic acid bacteria are sufficiently removed. Therefore, the quality of the product formulated with the disrupted lactic acid bacteria cell wall is less likely to deteriorate compared to the conventional heat-treated bacterial cells or their processed products, and it also has excellent dispersibility. In addition, it can be applied regardless of the enzyme sensitivity of lactic acid bacteria. This disrupted lactic acid bacteria cell wall can be suitably used, for example, as a blending material for foods, pharmaceuticals, cosmetics, and the like.

Brief Description of the Drawings

[0021]

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Mode for Carrying Out the Invention

[0022] The lactic acid bacteria used in the present invention are not particularly limited in terms of their type. For example, Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus gasseri, Lactobacillus cremoris, Lactobacillus helveticus, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus yoghurti, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus johnsonii, Lactobacillus mali and other bacteria of the genus Lactobacillus, Streptococcus thermophilus and other bacteria of the genus Streptococcus, Lactococcus fermentum, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Lactococcus plantarum, Lactococcus raffinolactis, LactococcusBacteria of the genus Lactococcus such as Lactococcus raffinolactis, bacteria of the genus Enterococcus such as Enterococcus faecalis and Enterococcus faecium, bacteria of the genus Weissella such as Weissella confusa, Weissella paramesenteroides, and Weissella viridescens, bacteria of the genus Pediococcus such as Pediococcus pentosaceus, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium suis, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis, Bifidobacterium globosum, and other bacteria of the genus Bifidobacterium, etc. are mentioned. In particular, Weissella viridescens, Streptococcus thermophilus, Lactobacillus casei, Bifidobacterium breve, Lactobacillus acidophilusLactobacillus acidophilus), Lactobacillus plantarum, etc. are preferred. As for Weissella viridescens, Weissella viridescens YIT0248 strain (ATCC 12706) is preferred. As for Streptococcus thermophilus, Streptococcus thermophilus YIT2037 strain (ATCC 19258) is preferred. As for Lactobacillus casei, Lactobacillus casei YIT9029 strain (FERM BP-1366) is preferred. As for Bifidobacterium breve, Bifidobacterium breve YIT4065 strain (FERM BP-6223) is preferred. As for Lactobacillus acidophilus, Lactobacillus acidophilus YIT0070 strain (ATCC 4356) is preferred. As for Lactobacillus plantarum, Lactobacillus plantarum YIT0102 strain (ATCC 14917) is preferred. In the present specification, the term "lactic acid bacteria" includes bacteria of the type generally called bifidobacteria.

[0023] These lactic acid bacteria can increase the number of bacteria or store the bacterial cells by means of a culture method such as a medium according to the type of lactic acid bacteria used and a storage method. Also, it is not necessary for them to be live bacteria, and those provided as dead bacterial cells may be used. The lactic acid bacteria may be used alone or in combination of two or more kinds.

[0024] In the present invention, based on the above lactic acid bacteria, what has had the contents other than the cell wall removed, specifically, provides a cell wall disrupted product of lactic acid bacteria. As a means for its preparation, it can be obtained by disrupting lactic acid bacteria by physical or mechanical means or the like and then appropriately washing with a washing solution. As the washing solution, a hydrophilic solution such as water or a buffer solution may be used. The washing solution may contain a surfactant such as a polyoxyethylene sorbitan type such as Tween 20. By including a surfactant in the washing solution, the effect of removing proteins and the like is enhanced. Washing can be performed by appropriately mixing and stirring the washing solution with the disrupted cells, followed by solid-liquid separation by means such as centrifugation or membrane filtration and recovering the cell components in the solid phase. The lactic acid bacteria may be dried by a drying means such as freeze-drying or sterilized by a heat treatment or the like before the washing treatment, and may be arbitrarily pretreated as appropriate. Also, after washing, further washing, drying by a drying means such as freeze-drying, sterilization by a heat treatment or the like, or other treatments may be arbitrarily performed as appropriate. In addition, in order to make the obtained cell wall disrupted product into a form such as a powder, granule, paste, tablet, or liquid suspension form, it may be appropriately and arbitrarily mixed with other materials.

[0025] It is preferable to use amino acids, which are constituent components such as proteins, as an index for whether the contents other than the cell wall have been sufficiently removed. Specifically, it can be confirmed by hydrolyzing the raw material lactic acid bacteria or the obtained cell wall disrupted product with concentrated hydrochloric acid and then performing amino acid analysis. Such amino acid analysis is a technique well-known to those skilled in the art. For example, if the amount of amino acids other than the types that constitute peptidoglycan or the like firmly bound to the cell wall of lactic acid bacteria is sufficiently reduced compared to the amount originally contained in the raw material lactic acid bacteria, it is a good indicator that the contents other than the cell wall have been sufficiently removed.

[0026] Specifically, taking the content of the amino acid contained in the raw material lactic acid bacteria reduced to, for example, 1 / 3 or less, or, for example, 1 / 5 or less, or in some cases, for example, 1 / 10 or less in terms of the unit of molality (mol / g) as an index is possible, and such numerical settings and the selection of amino acid types are arbitrary. As the amino acid used as an index, one type may be selected to determine whether the content satisfies the above conditions, or two or more types may be selected to determine whether the content of each amino acid satisfies the above conditions. Further, it may also be determined whether the total content of each amino acid satisfies the above conditions. The setting of the type of amino acid used as an index is also arbitrary. For example, amino acids such as proline (Pro), arginine (Arg), threonine (Thr), and phenylalanine (Phe) are often not firmly present in the cell walls of peptidoglycan and other lactic acid bacteria, so they can be preferably exemplified as amino acids serving as an index for sufficient removal of the contents other than the cell wall.

[0027] In addition, in other embodiments, whether the contents other than the cell wall have been sufficiently removed may be evaluated by the ratio of the amino acids to be removed to the components firmly present in the cell walls of peptidoglycan and other lactic acid bacteria. For example, but not limited to this, the total value of the contents of amino acids such as proline (Pro), arginine (Arg), threonine (Thr), and phenylalanine (Phe), which are amino acids other than the constituent amino acids of peptidoglycan, is reduced to 1 / 3 or less, or, for example, 1 / 5 or less, or in some cases, for example, 1 / 10 or less of the total value of the contents of amino acids such as serine (Ser), glutamic acid (Glu), and alanine (Ala), which are the constituent amino acids of peptidoglycan, can be used as an index, and such numerical settings and the selection of amino acid types are arbitrary.

[0028] In the present invention, in a more preferred embodiment, it is preferable that the disruption of lactic acid bacteria is carried out by wet means. That is, by disrupting in a state containing a solvent which is the elution destination of the content to be removed from the lactic acid bacteria, it is possible to more efficiently remove the content other than the cell wall. As such a disruption solvent for wet disruption, those equivalent to the cleaning liquid described above are preferably exemplified. That is, hydrophilic solutions such as water and buffer solutions may be used. The disruption solvent may contain a surfactant such as polyoxyethylene sorbitan type such as Tween 20. By including a surfactant in the disruption solvent, the effect of removing proteins and the like is enhanced. As means for wet disruption, for example, wet atomization means such as a wet atomization device ("Starburst Mini HJP-25001", Sugino Machine Ltd.) can be preferably exemplified. According to this type of device, a lactic acid bacteria sample suspended in a disruption solvent is introduced into a chamber maintained at a predetermined pressure state, whereby the lactic acid bacteria are disrupted and micronized, so that the content other than the cell wall can be removed more efficiently and the contamination is less. The degree of micronization is not particularly limited, but for example, when measuring the particle size distribution using a laser diffraction / scattering type particle size distribution measuring device or the like, it is preferably a particle size with a median diameter of 0.1 μm or more and 0.5 μm or less, and more preferably a particle size with a median diameter of 0.15 μm or more and 0.4 μm or less. Further, when the uniformity of the degree of micronization is represented by the standard deviation, it is preferably 0.25 μm or less, and more preferably 0.20 μm or less. Further, as the pressure condition for disruption by wet atomization means, it is preferably 100 MPa or more and 250 MPa or less, and more preferably 140 MPa or more and 250 MPa or less.

[0029] The disrupted lactic acid bacteria cell wall provided by the present invention can be suitably used in foods, pharmaceuticals, cosmetics, supplements, animal feeds, etc. In particular, for example, as the form of cosmetics, emulsions, creams, cleansings, massages, sunscreens, makeup bases, cream foundations, etc. can be mentioned. In addition, in this specification, "cosmetics" means including pharmaceuticals, quasi-drugs, and cosmetics as defined by laws regarding the assurance of quality, effectiveness, and safety of pharmaceuticals, medical devices, etc.

[0030] The content of the crushed lactic acid bacteria cell wall provided by the present invention in the above usage form is, for example, in the form of cosmetics, preferably 0.001% by mass or more and 10% by mass or less, more preferably 0.001% by mass or more and 5% by mass or less, still more preferably 0.01% by mass or more and 3% by mass or less, and particularly preferably 0.01% by mass or more and 1% by mass or less.

Examples

[0031] The present invention will be described in more detail with reference to the following examples, but these examples do not limit the scope of the present invention.

[0032] [Test Example 1] The properties of the micronized cells were verified. As test samples therefor, Preparation Examples 1-1 to 1-3 shown below were prepared.

[0033] <Preparation Example 1-1> (Heat treatment) Using Weissella viridescens YIT 0248 as the lactic acid bacterium, after culturing in Lactobacilli MRS broth medium (Becton Dickinson) according to a conventional method, it was centrifuged and washed with RO water. The cells were resuspended in sterilized ultrapure water and heat-treated (100 ° C, 30 minutes), and then freeze-dried.

[0034] <Preparation Example 1-2> (Micronization treatment, 5-pass condition) The heat-treated cells of Weissella viridescens YIT 0248 prepared in Preparation Example 1-1 were treated with 0.1% Tween-20, 4 mM MgCl 2It was suspended at a concentration of 2 mg / mL in 50 mM Tris-Maleate buffer (pH 7.0), and using a wet micronization device (“Starburst Mini HJP-25001”, manufactured by Sugino Machine), it was micronized by applying it to the device 5 times continuously under a pressure condition of 245 MPa. After micronization, SDS (sodium dodecyl sulfate) was added to a final concentration of 1 w / v%, and after sufficient stirring with a vortex, it was centrifuged at 100,000 rpm for 10 minutes. The supernatant was removed (a part of the supernatant was collected for measurement of the protein content), the precipitate was suspended in ultrapure water, and then centrifuged again at 100,000 rpm for 10 minutes, and the supernatant was removed. This washing operation was performed 3 times, and the final precipitate was lyophilized.

[0035] <Preparation Example 1-3> (Micronization treatment, 10-pass condition) The micronization treatment of the heat-treated cells of Weissella viridescens YIT 0248 prepared in Preparation Example 1-1 was carried out in the same manner as in Preparation Example 1-2, except that the number of times applied to the wet micronization device was 10 times.

[0036] (1) Measurement of protein content in supernatant Protein quantification was performed on the supernatant of the suspension of the lyophilized heat-treated cells in Preparation Example 1-1, or the supernatant of the first centrifugal washing after micronization in Preparation Examples 1-2 and 3, using a BCA protein assay kit (manufactured by Thermo Scientific).

[0037] As a result, as shown in Figure 1, compared to the supernatant of the suspension of the heat-treated cells before being applied to the wet micronization device, after micronization, a larger amount of protein was eluted on the supernatant side.

[0038] (2) Measurement of particle size distribution The test sample was suspended at a concentration of 50 μg / mL in purified water, and the particle size distribution was measured using a laser diffraction / scattering particle size distribution measuring device (LA-960, manufactured by HORIBA) with a batch cell.

[0039] As a result, as shown in Fig. 2, compared with the heat-treated cells before being subjected to the wet atomization device, after atomization, a decrease in the peak particle size corresponding to the number of treatments applied to the device and a uniformization (narrowing of the distribution width) were observed.

[0040] (3) Analysis of Amino Acid Composition The test sample was suspended in ultrapure water at a concentration of 1 mg / mL. 100 μL of the suspension was taken into a 16.5 mm screw-top test tube, 900 μL of HCl (6N) was added, and then the gas layer was replaced with argon. After heating the screw-top test tube at 100 °C for 18 hours, the solvent was evaporated using a centrifugal evaporator compatible with HCl to obtain a hydrolyzate.

[0041] The hydrolyzate was dissolved in ultrapure water to a concentration of 0.5 mg / mL to prepare an analysis sample. According to the protocol of the "AccQ Tag Ultra Drivatization Kit" (Waters), derivatization of amino acids was performed, and HPLC measurement was carried out under the following conditions. · Column: InertSustain (registered trademark) C18 (2 μm, 3 × 100 mm) · Column temperature: 50 °C · Flow rate: 0.45 mL / min. · Injection volume: 1.0 μL · Detection wavelength: 260 nm (PDA) · Mobile phase A: 0.1% formic acid / acetonitrile · Mobile phase B: 25 mM ammonium formate (pH 2.7) · Gradient: Table 1

[0042]

Table 1

[0043] As for the amino acids, standard products were prepared for 17 types of histidine (His), arginine (Arg), serine (Ser), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), threonine (Thr), alanine (Ala), proline (Pro), tyrosine (Tyr), methionine (Met), valine (Val), isoleucine (Ile), leucine (Leu), phenylalanine (Phe), cysteine (Cys), and lysine (Lys) to create a calibration curve, and the amount of each amino acid was determined. For cysteine (Cyc) and lysine (Lys), since a mixed peak with the reagent components for amino acid derivatization was detected, quantification became impossible.

[0044] An example of the chromatogram in HPLC analysis is shown in Figure 3, and the results of the amino acid quantification values are shown in Figure 4, respectively.

[0045] As shown in Figures 3 and 4, in the case of the cells treated only by heat treatment (Preparation Example 1-1), all 15 types of amino acids were detected in a certain amount. However, in the cells after 5 consecutive passes through the wet micronization device (Preparation Example 1-2) and the cells after 10 consecutive passes (Preparation Example 1-3), serine (Ser), glutamic acid (Glu), and alanine (Ala), which are the constituent amino acids of the peptidoglycan of Weissella viridescens YIT 0248, occupied most of the peaks (peaks 3, 6, and 8 in Figure 3). On the other hand, the amounts of amino acids other than the above types became lower due to the treatment with the wet micronization device.

[0046] [Test Example 2] The properties of the micronized cells were verified. For this purpose, Preparation Examples 2-1 to 2-3 shown below were prepared as test samples.

[0047] <Preparation Example 2-1> (Heat treatment) Streptococcus thermophilus (Streptococcus thermophilus YIT 2037) was used as the lactic acid bacterium. After culturing in Lactose-ILS medium according to a conventional method, it was centrifuged and washed with RO water. The cells were resuspended in sterilized ultrapure water, heat-treated (100 °C, 30 minutes), and then freeze-dried.

[0048] <Preparation Example 2-2> (5-pass condition for micronization treatment) The heat-treated cells of Streptococcus thermophilus (Streptococcus thermophilus YIT 2037) prepared in Preparation Example 2-1 were suspended at a concentration of 2 mg / mL in 50 mM Tris-Maleate buffer (pH 7.0) containing 0.1% Tween-20 and 4 mM MgCl 2 and micronized by continuously passing through the wet micronization device ("Starburst Mini HJP-25001", manufactured by Sugino Machine Limited) 5 times under a pressure condition of 245 MPa. After micronization, SDS was added to a final concentration of 1 w / v%, and after sufficient stirring with a vortex, it was centrifuged at 100,000 rpm for 10 minutes. The supernatant was removed (a part of the supernatant was collected for protein content measurement), the precipitate was suspended in ultrapure water, and then centrifuged again at 100,000 rpm for 10 minutes to remove the supernatant. This washing operation was performed 3 times, and the final precipitate was freeze-dried.

[0049] <Preparation Example 2-3> (10-pass condition for micronization treatment) The heat-treated cells of Streptococcus thermophilus (Streptococcus thermophilus YIT 2037) prepared in Preparation Example 2-1 were micronized in the same manner as in Preparation Example 2-2, except that the number of passes through the wet micronization device was 10 times.

[0050] (1) Measurement of protein content in the supernatant Protein quantification was performed on the supernatant of the suspension of the freeze-dried heat-treated cells in Preparation Example 2-1, or the supernatant of the first centrifugal washing after micronization in Preparation Examples 2-2 and 2-3, using a BCA Protein Assay Kit (Thermo Scientific).

[0051] As a result, as shown in Fig. 5, compared with the supernatant of the suspension of heat-treated cells before being subjected to the wet micronization device, more protein was eluted on the supernatant side after micronization.

[0052] (2) Measurement of particle size distribution The test sample was suspended in purified water at a concentration of 50 μg / mL, and the particle size distribution was measured using a batch cell with a laser diffraction / scattering particle size distribution analyzer (LA-960, HORIBA).

[0053] As a result, as shown in Fig. 6, compared with the heat-treated cells before being subjected to the wet micronization device, after micronization, a decrease in the peak particle size and a narrowing (reduction in the distribution width) corresponding to the number of passes through the device were observed.

[0054] (3) Analysis of amino acid composition The amino acid composition was analyzed in the same manner as in Test Example 1.

[0055] An example of the chromatogram in HPLC analysis is shown in Fig. 7, and the results of the amino acid quantification values are shown in Fig. 8, respectively.

[0056] As shown in Figs. 7 and 8, in the case of cells treated only by heat treatment (Preparation Example 2-1), all 15 types of amino acids were detected in a certain amount. However, in the cells after 5 consecutive passes through the wet micronization device (Preparation Example 2-2) and the cells after 10 consecutive passes (Preparation Example 2-3), glutamic acid (Glu) and alanine (Ala), which are the constituent amino acids of the peptidoglycan of Streptococcus thermophilus YIT 2037, occupied most of the content (peaks 6 and 8 in Fig. 7). On the other hand, the amounts of amino acids of other types became lower values due to the treatment with the wet micronization device.

[0057] [Test Example 3] The properties of the micronized cells were verified. For this purpose, Preparation Examples 3-1 to 3-2 shown below were prepared as test samples.

[0058] <Preparation Example 3-1> (Heat Treatment) Using Lactobacillus casei YIT 9029 as the lactic acid bacterium, after culturing in Lactose-ILS medium according to a conventional method, it was centrifuged and washed with RO water. The cells were resuspended in sterilized ultrapure water, heat-treated (100 °C, 30 minutes), and then freeze-dried.

[0059] <Preparation Example 3-2> (Micronization Treatment, 5-Pass Condition) The heat-treated cells of Lactobacillus casei YIT 9029 prepared in Preparation Example 3-1 were suspended at a concentration of 2 mg / mL in 50 mM Tris-Maleate buffer (pH 7.0) containing 0.1% Tween-20 and 4 mM MgCl 2 and were passed through the wet micronization device ("Starburst Mini HJP-25001", manufactured by Sugino Machine Limited) 5 times continuously under a pressure condition of 245 MPa for micronization. After micronization, SDS was added to a final concentration of 1 w / v%, and after sufficient stirring with a vortex, it was centrifuged at 100,000 rpm for 10 minutes. The supernatant was removed (a part of the supernatant was collected for the measurement of protein content), the precipitate was suspended in ultrapure water, and then centrifuged again at 100,000 rpm for 10 minutes to remove the supernatant. This washing operation was performed 3 times, and the final precipitate was freeze-dried.

[0060] (1) Measurement of Particle Size Distribution The test sample was suspended in purified water at a concentration of 50 μg / mL, and the particle size distribution was measured using a batch cell with a laser diffraction / scattering particle size distribution analyzer (LA-960, HORIBA).

[0061] As a result, as shown in Figure 9, a decrease in the peak particle size was observed after micronization compared to the heat-treated cells before passing through the wet micronization device.

[0062] (2) Analysis of Amino Acid Composition The amino acid composition was analyzed in the same manner as in Test Example 1.

[0063] An example of a chromatogram in HPLC analysis is shown in Fig. 10, and the results of amino acid quantification values are shown in Fig. 11, respectively.

[0064] As shown in Figs. 10 and 11, in the cells treated only by heat treatment (Preparation Example 3-1), all 15 kinds of amino acids were detected in a certain amount. However, in the cells after passing through the wet atomization device continuously 5 times (Preparation Example 3-2), aspartic acid (Asp), glutamic acid (Glu), and alanine (Ala), which are the constituent amino acids of peptidoglycan of Lactobacillus casei YIT 9029, occupied most of them (peaks 5, 6, and 8 in Fig. 10). On the other hand, the amounts of amino acids of other types became lower values due to the treatment in the wet atomization device.

[0065] [Test Example 4] The properties of the micronized cells were verified. As test samples for this purpose, Preparation Examples 4-1 to 4-2 shown below were prepared.

[0066] <Preparation Example 4-1> (Heat treatment) As the lactic acid bacterium, Bifidobacterium breve YIT 4065 was used. After anaerobic culture in Lactose-ILS medium according to a conventional method, it was centrifugally washed with RO water. The cells were resuspended in sterilized ultrapure water again, heat-treated (100 °C, 30 minutes), and then freeze-dried.

[0067] <Preparation Example 4-2> (Atomization treatment 5-pass condition) The heat-treated cells of Bifidobacterium breve YIT 4065 prepared in Preparation Example 4-1 were treated with 0.1% Tween-20, 4 mM MgCl 2It was suspended at a concentration of 2 mg / mL in 50 mM Tris-Maleate buffer (pH 7.0), and using a wet micronization device (“Starburst Mini HJP-25001”, Sugino Machine), it was continuously applied to the device 5 times under pressure conditions of 245 MPa for micronization. After micronization, SDS was added to a final concentration of 1 w / v%, and after sufficient stirring with a vortex, it was centrifuged at 100,000 rpm for 10 minutes. The supernatant was removed (a part of the supernatant was collected for protein content measurement), and the precipitate was suspended in ultrapure water and then centrifuged again at 100,000 rpm for 10 minutes to remove the supernatant. This washing operation was performed 3 times, and the final precipitate was lyophilized.

[0068] (1) Measurement of particle size distribution The test sample was suspended in purified water at a concentration of 50 μg / mL, and the particle size distribution was measured using a laser diffraction / scattering particle size distribution analyzer (LA-960, HORIBA) with a batch cell.

[0069] As a result, as shown in Figure 12, compared with the heat-treated cells before being applied to the wet micronization device, after micronization, a decrease in peak particle size and homogenization (narrowing of the distribution width) were observed.

[0070] (2) Analysis of amino acid composition The amino acid composition was analyzed in the same manner as in Test Example 1.

[0071] An example of the chromatogram in HPLC analysis is shown in Figure 13, and the results of the amino acid quantification values are shown in Figure 14, respectively.

[0072] As shown in FIGS. 13 and 14, in the case of the cells treated only by heat treatment (Preparation Example 4-1), all 15 types of amino acids were detected in a certain amount. However, in the case of the cells after passing through the wet atomization device 5 times continuously (Preparation Example 4-2), glycine (Gly), glutamic acid (Glu), and alanine (Ala), which are the constituent amino acids of the peptidoglycan of Bifidobacterium breve YIT 4065, occupied most of the proportion (peaks 4, 6, and 8 in FIG. 10). On the other hand, the amounts of amino acids of types other than the above became lower values due to the treatment in the wet atomization device.

[0073] [Test Example 5] The properties of the micronized cells were verified. As test samples for this purpose, Preparation Examples 5-1 to 5-2 shown below were prepared.

[0074] <Preparation Example 5-1> (Heat treatment) Lactobacillus acidophilus YIT 0070 was used as the lactic acid bacterium. After culturing in MRS medium according to a conventional method, it was centrifuged and washed with RO water. The cells were resuspended in sterilized ultrapure water again, heat-treated (100 °C, 30 minutes), and then freeze-dried.

[0075] <Preparation Example 5-2> (Micronization treatment 5-pass condition) The heat-treated cells of Lactobacillus acidophilus YIT 0070 prepared in Preparation Example 5-1 were treated with 0.1% Tween-20, 4 mM MgCl 2It was suspended at a concentration of 2 mg / mL in 50 mM Tris-Maleate buffer (pH 7.0), and using a wet micronization device ("Starburst Mini HJP-25001", Sugino Machine), it was continuously applied to the device 5 times under a pressure condition of 245 MPa for micronization. After micronization, SDS was added to a final concentration of 1 w / v%, and after sufficient stirring with a vortex, it was centrifuged at 100,000 rpm for 10 minutes. The supernatant was removed (a part of the supernatant was collected for the measurement of protein content), and the precipitate was suspended in ultrapure water and then centrifuged again at 100,000 rpm for 10 minutes to remove the supernatant. This washing operation was performed 3 times, and the final precipitate was lyophilized.

[0076] (1) Measurement of particle size distribution The test sample was suspended in purified water at a concentration of 50 μg / mL, and the particle size distribution was measured using a laser diffraction / scattering particle size distribution analyzer (LA-960, HORIBA) with a batch cell.

[0077] As a result, as shown in Figure 15, compared with the heat-treated cells before being applied to the wet micronization device, after micronization, a decrease in peak particle size and homogenization (narrowing of the distribution width) were observed.

[0078] (2) Analysis of amino acid composition The amino acid composition was analyzed in the same manner as in Test Example 1.

[0079] An example of the chromatogram in HPLC analysis is shown in Figure 16, and the results of the amino acid quantification values are shown in Figure 17, respectively.

[0080] As shown in FIGS. 16 and 17, in the case of the cells only subjected to heat treatment (Preparation Example 5-1), all 15 types of amino acids were detected in a certain amount. However, in the case of the cells after passing through the wet atomization device 5 times continuously (Preparation Example 5-2), aspartic acid (Asp), glutamic acid (Glu), and alanine (Ala), which are the constituent amino acids of peptidoglycan of Lactobacillus acidophilus YIT 0070, occupied most (peaks 5, 6, and 8 in FIG. 16). On the other hand, the amounts of amino acids of types other than the above became lower values due to the treatment in the wet atomization device.

[0081] [Test Example 6] The properties of the micronized cells were verified. As test samples for this purpose, Preparation Examples 6-1 to 6-2 shown below were prepared.

[0082] <Preparation Example 6-1> (Heat treatment) Lactobacillus plantarum YIT 0102 was used as the lactic acid bacterium. After culturing in MRS medium according to a conventional method, it was centrifuged and washed with RO water. The cells were resuspended in sterilized ultrapure water and heat-treated (100 °C, 30 minutes), and then freeze-dried.

[0083] <Preparation Example 6-2> (5-pass micronization treatment conditions) The heat-treated cells of Lactobacillus plantarum YIT 0102 prepared in Preparation Example 6-1 were treated with 0.1% Tween-20, 4 mM MgCl 2It was suspended at a concentration of 2 mg / mL in 50 mM Tris-Maleate buffer (pH 7.0), and using a wet micronization device ("Starburst Mini HJP-25001", Sugino Machine), it was continuously applied to the device 5 times under pressure conditions of 245 MPa for micronization. After micronization, SDS was added to a final concentration of 1 w / v%, and after sufficient stirring with a vortex, it was centrifuged at 100,000 rpm for 10 minutes. The supernatant was removed (a part of the supernatant was collected for protein content measurement), the precipitate was suspended in ultrapure water, and then centrifuged again at 100,000 rpm for 10 minutes to remove the supernatant. This washing operation was performed 3 times, and the final precipitate was freeze-dried.

[0084] (1) Measurement of particle size distribution The test sample was suspended in purified water at a concentration of 50 μg / mL, and the particle size distribution was measured using a laser diffraction / scattering particle size distribution analyzer (LA-960, HORIBA) with a batch cell.

[0085] As a result, as shown in Figure 18, a decrease in the peak particle size was observed after micronization compared to the heat-treated cells before being applied to the wet micronization device.

[0086] (2) Analysis of amino acid composition The amino acid composition was analyzed in the same manner as in Test Example 1.

[0087] An example of the chromatogram in HPLC analysis is shown in Figure 19, and the results of the amino acid quantification values are shown in Figure 20, respectively.

[0088] As shown in FIGS. 19 and 20, in the cells treated only by heat treatment (Preparation Example 6-1), 14 types of amino acids were detected in a certain amount. However, in the cells after passing through the wet micronization device 5 times continuously (Preparation Example 6-2), glutamic acid (Glu) and alanine (Ala), which are the constituent amino acids of peptidoglycan of Lactobacillus plantarum YIT 0102, occupied most of the amount (Peaks 6 and 8 in FIG. 19). Note that the peak indicated by "◆" in FIG. 19 is the peak of meso-DAP (meso-diaminopimelic acid) that constitutes the peptidoglycan of Lactobacillus plantarum YIT 0102. On the other hand, the amounts of amino acids of other types became lower values due to the treatment in the wet micronization device.

[0089] [Test Example 7] The properties of the micronized cells were verified. As test samples for this purpose, Preparation Examples 7-1 to 7-6 shown below were prepared.

[0090] <Preparation Example 7-1> (Heat treatment) Weissella viridescens YIT 0248 was used as the lactic acid bacterium. After culturing in Lactobacilli MRS broth medium (Becton Dickinson) according to a conventional method, it was centrifugally washed with RO water. The cells were resuspended in sterilized ultrapure water and heat-treated (100 °C, 30 minutes). Then, SDS was added to a final concentration of 1 w / v%, and after sufficient stirring with a vortex, it was centrifuged at 100,000 rpm for 10 minutes. The supernatant was removed (a part of the supernatant was collected for the measurement of protein content), and the precipitate was resuspended in ultrapure water and then centrifuged again at 100,000 rpm for 10 minutes to remove the supernatant. This washing operation was performed 3 times, and the final precipitate was freeze-dried to obtain a powder.

[0091] <Preparation Example 7-2> (Micronization treatment) The heat-treated cells of Weissella viridescens YIT 0248 prepared in Preparation Example 1-1 were suspended at a concentration of 2 mg / mL in 50 mM Tris-Maleate buffer (pH 7.0) containing 0.1% Tween-20 and 4 mM MgCl 2 and were micronized by continuously passing them through a wet micronization device ("Starburst Mini HJP-25001", manufactured by Sugino Machine Limited) 10 times under a pressure condition of 245 MPa. After micronization, SDS was added to a final concentration of 1 w / v%, and after thoroughly stirring with a vortex, centrifugation was performed at 100,000 rpm for 10 minutes. The supernatant was removed (a part of the supernatant was collected for the measurement of protein content), the precipitate was suspended in ultrapure water, and then centrifugation was performed again at 100,000 rpm for 10 minutes to remove the supernatant. This washing operation was performed 3 times, and the final precipitate was lyophilized to obtain a powder.

[0092] <Preparation Example 7-3> (Enzyme Treatment) The heat-treated cells of Weissella viridescens YIT 0248 prepared in Preparation Example 1-1 were suspended at a concentration of 2 mg / mL in 50 mM Tris-Maleate buffer (pH 7.0) containing 0.1% Tween-20 and 4 mM MgCl 2 and were reacted with lysozyme at 50 μg / mL for 120 minutes at a temperature of 40°C. After the reaction, the enzyme activity was inactivated by heat treatment at 100°C for 15 minutes, and then lyophilized to obtain a powder.

[0093] <Preparation Example 7-4> (Heat Treatment Only) A lactic acid bacterium powder was prepared in the same manner as in Preparation Example 7-1, except that Streptococcus thermophilus YIT 2037 was used as the lactic acid bacterium.

[0094] <Preparation Example 7-5> (Micronization Treatment) A lactic acid bacterium powder was prepared in the same manner as in Preparation Example 7-2, except that Streptococcus thermophilus (Streptococcus thermophilus YIT 2037) was used as the lactic acid bacterium.

[0095] <Preparation Example 7-6> (Enzyme treatment) A lactic acid bacterium powder was prepared in the same manner as in Preparation Example 7-3, except that Streptococcus thermophilus (Streptococcus thermophilus YIT 2037) was used as the lactic acid bacterium.

[0096] (1) Stability evaluation in powder form The prepared lactic acid bacterium powder was stored at 5°C, and it was observed and evaluated whether moisture absorption, coagulation, and off-odor occurred. Specifically, on the confirmation day of the 7th day, its stability was evaluated according to the following evaluation criteria.

[0097] (Stability evaluation criteria) 〇; Good △; Slightly poor (slight moisture absorption, coagulation, or off-odor is observed) ×; Poor (moisture absorption, coagulation, or off-odor is observed)

[0098] The evaluation results are shown in Figure 21. In the enzyme-treated cells (Preparation Examples 7-3 and 7-6), coagulation due to moisture absorption was observed in both cases. On the other hand, in the micronized cells mainly containing only the cell wall (Preparation Examples 7-2 and 7-5), neither moisture absorption nor coagulation was observed.

[0099] (2) Stability evaluation in suspension The prepared lactic acid bacterium powder was suspended in purified water at a concentration of 0.15 mg / mL, and it was observed and evaluated whether turbidity, discoloration, and off-odor occurred under each storage condition of 50°C, or 5°C: 84 hours and 50°C: 84 hours repeated (hereinafter simply referred to as "5°C 50°C repeat"). Specifically, after 3 months of storage, on the confirmation day of the 90th day, its stability was evaluated according to the following evaluation criteria.

[0100] (Stability evaluation criteria) 〇; Good △; Slightly defective (slight turbidity, discoloration, or abnormal odor is observed) ×; Defective (turbidity, discoloration, or abnormal odor is observed)

[0101] The evaluation results are shown in Fig. 22. For the cells treated only by heat treatment (Preparation Examples 7-1 and 7-4) and the cells treated by enzyme treatment (Preparation Examples 7-3 and 7-6), the stability evaluation results were defective or slightly defective under any storage conditions. On the other hand, for the micronized cells mainly composed of only cell walls (Preparation Examples 7-2 and 7-5), turbidity, discoloration, or abnormal odor was not observed, and the stability evaluation results were good under any storage conditions.

[0102] (3) Microscopic observation After the prepared lactic acid bacteria powder was fixed with glutaraldehyde and dried, it was platinum-coated and then observed with a desktop scanning electron microscope (objective magnification: 10,000 times).

[0103] Fig. 23 exemplarily shows the microscopic photographs taken for each test sample.

[0104] As shown in Fig. 23, for the cells treated only by heat treatment (Preparation Examples 7-1 and 7-4), no disruption of the cell wall was observed, and a capsule-like three-dimensional structure was observed. Also, for the cells treated by enzyme treatment, in the case of Streptococcus thermophilus with low lysozyme sensitivity (Preparation Example 7-6), no disruption of the cell wall was observed, and a capsule-like three-dimensional structure was observed, but in the case of Weissella viridescens with high lysozyme sensitivity (Preparation Example 7-3), the capsule-like three-dimensional structure of the cell wall disappeared, and an aggregate of disrupted cell walls was observed. On the other hand, for the micronized cells (Preparation Examples 7-2 and 7-5), the capsule-like three-dimensional structure of the cell wall disappeared in both lactic acid bacteria, and an aggregate of disrupted cell walls was observed.

[0105] (4) Dispersibility evaluation When the prepared lactic acid bacteria powder was suspended in purified water at a concentration of 0.15 mg / mL, most of the cells treated only with heat treatment (Preparation Examples 7-1 and 7-4) precipitated after standing. On the other hand, in the case of the micronized cells (Preparation Examples 7-2 and 7-5) and the enzyme-treated cells (Preparation Examples 7-3 and 7-6), some precipitation was observed after standing, but the dispersibility was better than that of the cells treated only with heat treatment (Preparation Examples 7-1 and 7-4).

[0106] [Strain] · Weissella viridescens YIT0248 strain (ATCC 12706) · Streptococcus thermophilus YIT2037 strain (ATCC 19258) · Lactobacillus casei YIT9029 strain (FERM BP-1366) · Bifidobacterium breve YIT4065 strain (FERM BP-6223) · Lactobacillus acidophilus YIT0070 strain (ATCC 4356) · Lactobacillus plantarum YIT0102 strain (ATCC 14917)

Claims

Claim 1 A method for producing a lactic acid bacteria cell wall disruptant (excluding those prepared through treatment with a cell wall degrading enzyme or a proteolytic enzyme) by disrupting lactic acid bacteria using wet micronization means and performing solid-liquid separation to recover the solid phase thereof, comprising: the disruption by the wet micronization means is such that the pressure condition in the chamber into which the lactic acid bacteria are introduced is 100 MPa or more and 250 MPa or less, and the treatment by the means is repeated a plurality of times; the disruption preparation by the wet micronization means to be subjected to the solid-liquid separation is one that has been subjected to a washing treatment with a washing liquid after the disruption by the wet micronization means, the method for producing the lactic acid bacteria cell wall disruptant. Claim 2 The method for producing a lactic acid bacteria cell wall disruptant according to claim 1, wherein the lactic acid bacteria to be subjected to the disruption by the wet micronization means are prepared as a sample in a solution containing a buffer and / or a surfactant. Claim 3 The method for producing a lactic acid bacteria cell wall disruptant according to claim 1 or 2, wherein the disruption preparation by the wet micronization means to be subjected to the solid-liquid separation is one that has been subjected to a plurality of washing treatments with a washing liquid after the disruption by the wet micronization means.

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