Method for producing cell wall polysaccharide-containing preparation and cell wall polysaccharide-containing preparation

Abstract

To provide a method for preparing high-molecular-weight polysaccharides derived from the cell walls of lactic acid bacteria with highly purity, and a preparation containing high-molecular-weight polysaccharides thus obtained with high purity.SOLUTION: A method for producing a cell wall polysaccharide-containing preparation includes the steps of: preparing a polysaccharide extract by extracting cell wall polysaccharides from lactic acid bacteria; subjecting the polysaccharide extract to an ultrafiltration membrane; and recovering the target polysaccharide with a molecular weight exceeding the nominal molecular weight cutoff of the ultrafiltration membrane from the filtrate having passed through the ultrafiltration membrane.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a technique for preparing polysaccharides derived from the cell walls of lactic acid bacteria. [Background technology]

[0002] In recent years, attempts have been made to utilize components derived from microbial cells as functional components for the living body. For example, Patent Document 1 discloses an invention of an antiulcer agent containing lactic acid bacteria cells as an active ingredient. Also, for example, Patent Document 2 discloses an invention of an anti-HIV agent containing lactic acid bacteria cells as an active ingredient. Also, for example, Patent Documents 3 and 4 disclose that cell wall-derived polysaccharides present in the cell walls of lactic acid bacteria have the functionality of regulating the production amounts of cytokines such as IL6 and IL12 produced by immune regulatory cells.

[0003] On the other hand, according to Non-Patent Documents 1 to 4, it is becoming clear that the molecular entity of the cell wall polysaccharides derived from lactic acid bacteria having such functionality is a high molecular weight polysaccharide having a molecular weight of more than 100,000. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 4-5236 [Patent Document 2] Japanese Patent Application Publication No. 4-145026 [Patent Document 3] JP 2003-73286 A [Patent Document 4] JP 2015-129114 A [Non-patent literature]

[0005] [Non-Patent Document 1] Structure of Polysaccharide-Peptidoglycan Complex from the Cell Wall of Lactobacillus Casei YIT9018, J. Chem. Biochemistry (1990), 108(4), p568-571.

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[0006] Conventionally, preparations containing polysaccharides derived from the cell walls of lactic acid bacteria have been prepared by extracting cell wall polysaccharides from lactic acid bacteria to obtain a polysaccharide extract, and then applying the polysaccharide extract to a predetermined molecular weight fractionation means such as a dialysis membrane, an ultrafiltration membrane, gel filtration, etc. However, it has sometimes been difficult to separate and prepare high molecular weight polysaccharides, such as those having a molecular weight of more than 100,000, from impurities having similar molecular weights.

[0007] On the other hand, if the preparation from lactic acid bacteria is in a more purified form, it will be useful as a standard product for research, and it is also desirable from the viewpoint of expanding the range of applications to materials to be incorporated into pharmaceuticals, foods, cosmetics, etc.

[0008] An object of the present invention is to provide a method for preparing a highly purified high molecular weight polysaccharide derived from the cell walls of lactic acid bacteria, and to provide a preparation containing the highly purified high molecular weight polysaccharide thus obtained. [Means for solving the problem]

[0009] As a result of intensive research to achieve the above-mentioned object, the inventors have found that some polysaccharides derived from the cell walls of lactic acid bacteria have a molecular weight of more than 100,000, but can pass through ultrafiltration membranes with a nominal molecular weight cut-off smaller than that, and have thus completed the present invention.

[0010] That is, in a first aspect, the present invention provides a method for producing a cell wall polysaccharide-containing preparation, comprising the steps of preparing a polysaccharide extract by extracting cell wall polysaccharides from lactic acid bacteria, subjecting the polysaccharide extract to an ultrafiltration membrane, and recovering a target polysaccharide having a molecular weight exceeding the nominal molecular cutoff of the ultrafiltration membrane from the liquid that has passed through the ultrafiltration membrane.

[0011] According to the present invention, by subjecting a polysaccharide extract obtained by extracting cell wall polysaccharides from lactic acid bacteria to an ultrafiltration membrane, it is possible to remove molecules that do not pass through the ultrafiltration membrane. Polysaccharides that have a molecular weight exceeding the nominal molecular cutoff of the ultrafiltration membrane used but are structurally capable of passing through the ultrafiltration membrane can be separated from impurities that cannot pass through, and recovered in the permeate that has passed through the ultrafiltration membrane.

[0012] In the production method according to the first aspect of the present invention, the permeate that has passed through the ultrafiltration membrane may be subjected to further molecular weight fractionation means to purify and recover the target polysaccharide. In this manner, the permeate that has passed through the ultrafiltration membrane is purified by further molecular weight fractionation means, so that the target polysaccharide can be obtained with a higher degree of purification.

[0013] In the production method according to the first aspect of the present invention, the permeate that has passed through the ultrafiltration membrane may be further subjected to an additional ultrafiltration membrane having a nominal molecular weight cutoff less than the nominal molecular weight cutoff of the ultrafiltration membrane, and the target polysaccharide may be recovered in the retentate that does not pass through the additional ultrafiltration membrane. In this way, the permeate that has passed through the ultrafiltration membrane is further subjected to an ultrafiltration membrane having a smaller nominal molecular weight cutoff to obtain the retentate, which makes it possible to remove molecules that pass through the additional ultrafiltration membrane, and to obtain the target polysaccharide with a higher degree of purification.

[0014] In the production method according to the first aspect of the present invention, the permeate that has passed through the ultrafiltration membrane may be further subjected to column chromatography to recover the target polysaccharide in the eluted fraction, whereby the permeate that has passed through the ultrafiltration membrane is further subjected to a preparative column, thereby enabling the target polysaccharide to be obtained with a higher degree of purification.

[0015] The production method according to the first aspect of the present invention may further include a step of subjecting the extract from the lactic acid bacteria to a molecular weight fractionation means other than the ultrafiltration membrane before passing through the ultrafiltration membrane. In this way, the target polysaccharide can be obtained with a higher degree of purification by removing low molecular weight molecules in advance, for example, by dialysis or chromatography, before passing through the ultrafiltration membrane.

[0016] In the method according to the first aspect of the present invention, in the step of preparing the polysaccharide extract, a cell wall lytic enzyme may be used to extract cell wall polysaccharides from the lactic acid bacteria, which allows more effective separation of polysaccharides from the lactic acid bacteria, thereby enabling more efficient production of a preparation containing the desired polysaccharides.

[0017] On the other hand, in a second aspect, the present invention provides a cell wall polysaccharide-containing preparation comprising a polysaccharide derived from a lactic acid bacteria cell wall, the polysaccharide passing through an ultrafiltration membrane with a nominal molecular weight cutoff of 30,000 and having a molecular weight of 100,000 or more as estimated from a chromatogram obtained by a gel filtration column and differential refractive index detection.

[0018] In the cell wall polysaccharide-containing preparation according to the second aspect of the present invention, the polysaccharide may be derived from Lacticaseibacillus paracasei.

[0019] In the cell wall polysaccharide-containing preparation according to the second aspect of the present invention, the polysaccharide may be formed by the cps1 gene cluster of Lacticaseibacillus paracasei. Effect of the Invention

[0020] According to the present invention, high molecular weight polysaccharides derived from the cell walls of lactic acid bacteria can be prepared with a high degree of purification. In addition, a preparation containing the high molecular weight polysaccharides obtained with such a high degree of purification can be provided. This cell wall polysaccharide-containing preparation is useful as a standard for research, or can be suitably used as a material to be incorporated into medicines, foods, cosmetics, etc. [Brief description of the drawings]

[0021] [Figure 1] 1 is a table showing examples of images of the YIT 9029 strain and the Δcps strain cells Gram-stained and observed under a microscope in Test Example 1. [Diagram 2] 1 is a chart showing an example of an image observed under a fluorescent microscope when the antibody reactivity of cells of the YIT 9029 strain and the Δcps strain was examined in Test Example 2. [Diagram 3] 1 is a graph showing the results of separation and analysis by gel filtration column chromatography of the total cell wall polysaccharide solution before ultrafiltration, and the retentate and permeate after ultrafiltration in Test Example 3. [Figure 4] 1 is a graph showing the results of separation and analysis by gel filtration column chromatography of the total cell wall polysaccharide solution before ultrafiltration, and the retentate and permeate after ultrafiltration in Test Example 4. [Diagram 5] 1 is a graph showing the results of separation and analysis by gel filtration column chromatography of the total cell wall polysaccharide solution before ultrafiltration, and the retentate and permeate after ultrafiltration in Test Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In the present invention, first, a polysaccharide extract is prepared by extracting cell wall polysaccharides from lactic acid bacteria.

[0023] The lactic acid bacteria is not particularly limited, and examples thereof include bacteria of the genus Lacticaseibacillus, such as Lacticaseibacillus paracasei and Lacticaseibacillus casei, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus cremoris, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. Lactobacillus bacteria such as Lactobacillus delbrueckii and Lactobacillus johnsonii, Ligilactobacillus bacteria such as Ligilactobacillus salivarius, Limosilactobacillus bacteria such as Limosilactobacillus fermentum, Liquorilactobacillus bacteria such as Liquorilactobacillus mali, Lactiplantibacillus bacteria such as Lactiplantibacillus plantarum, Streptococcus bacteria such as Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Lactococcus lactis Lactococcus lactis subsp.Lactococcus bacteria such as Lactococcus cremoris, Lactococcus plantarum, and Lactococcus raffinolactis, Enterococcus bacteria such as Enterococcus faecalis and Enterococcus faecium, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium suis, and Bifidobacterium infantis. Examples of the lactic acid bacteria include Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis, and Bifidobacterium globosum. In this specification, the term "lactic acid bacteria" includes the type of bacteria generally called bifidobacteria. In addition, one type of lactic acid bacteria may be used alone, or two or more types may be used in combination.

[0024] Lactic acid bacteria preferably used in the present invention include, but are not limited to, Lacticaseibacillus paracasei. For example, Lacticaseibacillus paracasei YIT 9029 strain (old classification: Lactobacillus casei YIT 9029 strain) is internationally deposited as Lactobacillus casei YIT 9029 (FERM BP-1366, date of deposit: May 1, 1981) at the National Institute of Technology and Evaluation, International Patent Organism Depositary Center (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818), which is the international depositary authority of the Budapest Treaty.

[0025] In recent years, lactic acid bacteria of the genus Lactobacillus have been reclassified. That is, the genus of lactic acid bacteria that previously belonged to the genus Lactobacillus has been subdivided, and the genus names of some of the species have been changed.

[0026] (Reclassification of lactic acid bacteria) ·Zheng et al., A taxonomic note on the genus Lactobacillus : Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int. J. Syst. Evol. Microbiol. 2020 Apr; 70(4):2782-2858DOI 10.1099 / ijsem.0.004107

[0027] Therefore, in this specification, the lactic acid bacteria are indicated by the new classification after the reclassification. For example, among the lactic acid bacteria classified as Lactobacillus casei or Lactobacillus paracasei in the old classification, those that can be newly classified as Lacticaseibacillus paracasei are included in Lacticaseibacillus paracasei in this application.

[0028] Since suitable media, culture methods, and preservation methods are known for each type of lactic acid bacteria, the bacteria can be multiplied and maintained according to such known methods. For example, preferred culture media for Lacticaseibacillus paracasei include Rogosa medium (Rogosa et al., A Selective Medium for the Isolation and Enumeration of Oral Lactobacilli. Journal of Dental Research, 1951 Oct; 30(5): 682-689. doi:10.1177 / 00220345510300051201) and de Man Rogosa Sharpe (MRS) medium (de Man et al., A Medium for the Cultivation of Lactobacilli. Journal of Applied Bacteriology. 1960 Apr; 23(1): 130-135. doi:10.1111 / j.1365-2672.1960.tb00188.x), milk, skim milk, soy milk, fruit juice, vegetable juice, etc. Commercially available products include "Difco Lactobacilli MRS broth" (Becton, Dickinson and Company), "GAM bouillon" (Nissui Pharmaceutical Co., Ltd.), "modified GAM bouillon" (Nissui Pharmaceutical Co., Ltd.), "MRS broth" (Merck), etc. Cultivation can be performed by inoculating the bacteria in a liquid medium and culturing them under conditions of 30 to 42 ° C., or by applying the bacteria to a plate medium and culturing them under similar temperature conditions. After culturing in a liquid medium, the lactic acid bacteria can be collected by removing the culture supernatant by centrifugation or the like. The collected bacteria can be suspended in fresh water or an aqueous buffer solution, and the supernatant can be removed by centrifugation or the like, to wash the bacteria. The lactic acid bacteria may be subjected to the cell wall polysaccharide extraction procedure described below in a live state, or may be subjected to the extraction procedure described below in a state in which they have been killed by heat treatment or the like, or in a freeze-dried state.

[0029] The method for extracting cell wall polysaccharides from lactic acid bacteria is not particularly limited as long as it can liberate polysaccharides constituting the cell wall of lactic acid bacteria in the obtained extract. For example, a method such as hot water extraction can be used conveniently. For example, the lactic acid bacteria cells obtained by appropriate culture are washed with water or an aqueous buffer solution, suspended in fresh water or an aqueous buffer solution, and heated for example at 80 to 121°C for 10 to 120 minutes by autoclaving, and then centrifuged to remove solid cell debris, and a polysaccharide extract containing high molecular weight polysaccharides liberated from the lactic acid bacteria cell wall is obtained as the supernatant.

[0030] On the other hand, so-called cell wall lytic enzymes are known to have the activity of acting on peptidoglycan constituting the cell wall of lactic acid bacteria and partially decomposing it, and thus can effectively liberate polysaccharides derived from the cell wall of lactic acid bacteria, so that treatment with such cell wall lytic enzymes may be used as a means for extracting cell wall polysaccharides from lactic acid bacteria.

[0031] The treatment with the cell wall-lytic enzyme may be carried out, for example, by the method described in JP-A-4-5236 and JP-A-2003-73286. More specifically, first, the lactic acid bacteria cells are suspended in an isotonic solution and treated with a cell wall-lytic enzyme such as N-acetylmuramidase, lysozyme, labiase, achromopeptidase, etc. Prior to this treatment, the cells may be disrupted by ultrasonic treatment, French press, etc. Solid cell debris is removed from the cell suspension after the enzyme treatment by centrifugation, and a polysaccharide extract containing high molecular weight polysaccharides released from the lactic acid bacteria cell walls is obtained as the supernatant.

[0032] The extract from the lactic acid bacteria may be treated with a nuclease or a protease, if necessary, to reduce the molecular weight of the components contained as high molecular weight substances. The extract from the lactic acid bacteria may also be subjected to treatments such as dialysis, ultrafiltration, gel filtration, desalting, and ion removal, if necessary.

[0033] In any embodiment not limited by the present invention, the extract from lactic acid bacteria is preferably subjected to molecular weight fractionation means such as dialysis, chromatography, ultrafiltration, gel filtration, density gradient centrifugation, etc. before processing with an ultrafiltration membrane described later. This makes it possible to remove unnecessary substances such as low molecular weight polysaccharides, nucleic acid decomposition products, protein decomposition products, oligosaccharides, polypeptides, enzymes, and salts. As a guide for fractionation, the molecular weight may be 100 or more, 500 or more, 1000 or more, 5000 or more, or 10000 or more. However, it is not necessarily necessary to carry out such molecular weight fractionation means for removing low molecular weight substances before processing with an ultrafiltration membrane described later. Alternatively, in some cases, it may be carried out after processing with an ultrafiltration membrane described later as necessary. In addition, other substances may be added as appropriate and arbitrary as long as there is no problem in ensuring the degree of purification of the desired high molecular weight polysaccharide. Examples of such substances include pH adjusters, salts, etc.

[0034] The polysaccharide extract containing the high molecular weight polysaccharides released from the lactic acid bacteria cell walls may be concentrated by evaporation or an acylation device, or freeze-dried, as necessary. In that case, when subjecting the extract to treatment using an ultrafiltration membrane as described below, the volume may be restored as appropriate with water or an aqueous buffer solution before treatment.

[0035] In the present invention, the polysaccharide extract prepared as described above is then subjected to an ultrafiltration membrane.

[0036] This treatment allows the retention of high molecular weight substances that do not pass through the ultrafiltration membrane in the retentate. Some polysaccharides derived from the cell walls of lactic acid bacteria have a molecular weight of over 100,000, but pass through ultrafiltration membranes with a nominal molecular weight cutoff smaller than that, so such high molecular weight polysaccharides can be transferred to the permeate.

[0037] The ultrafiltration membrane to be used may have, for example, a nominal molecular weight cutoff of 30,000, 50,000, 100,000, 150,000, 200,000, 250,000, or 300,000. Commercially available products include, for example, the "Amicon Ultra Centrifugal Ultrafiltration Filter" series (Merck) and the "Vivaspin" series (Cytiva). As the ultrafiltration membrane, a plurality of membranes having the desired nominal molecular weight cutoff may be used in combination, or the molecular weight cutoff size may be gradually changed until the membrane having the desired nominal molecular weight cutoff is finally used.

[0038] In the present invention, the target polysaccharide is finally recovered in the permeate obtained by subjecting the mixture to the ultrafiltration membrane as described above. Here, "recovering the target polysaccharide" refers to recovering the high molecular weight polysaccharide that has passed through the ultrafiltration membrane into the permeate separately from impurities that cannot pass into the permeate in this manner.

[0039] In an optional embodiment not limited to the present invention, the liquid passing through the ultrafiltration membrane may be subjected to further molecular weight fractionation to purify and recover the target polysaccharide.

[0040] The means for further molecular weight fractionation may be an additional ultrafiltration membrane having a nominal molecular weight cutoff less than the nominal molecular weight cutoff value of the ultrafiltration membrane used above. The ultrafiltration membrane used may have a nominal molecular weight cutoff of, for example, 2000, 3000, 5000, 10,000, 30,000, 50,000, or 100,000. As for commercially available products, as above, the "Amicon Ultra Centrifugal Ultrafiltration Filter" series (Merck) and the "Vivaspin" series (Cytiva) can be mentioned. That is, by subjecting the permeate through the above-mentioned ultrafiltration membrane to an additional ultrafiltration membrane and recovering the retentate, low molecular weight substances can be transferred to and removed from the permeate through the additional ultrafiltration membrane. As the additional ultrafiltration membrane, a plurality of membranes having the desired nominal molecular weight cutoff may be used in combination, or the molecular weight cutoff size may be gradually changed so that the membrane having the desired nominal molecular weight cutoff is finally used. For example, a preferred embodiment is one in which an ultrafiltration membrane is used while gradually reducing the size from 300,000 or more to 250,000, 200,000, 150,000, 100,000, 50,000, or 30,000, and the like, and the permeate is recovered, and finally, one having a nominal molecular weight cutoff of 10,000 is used.

[0041] Further molecular weight fractionation may be achieved by column chromatography such as gel filtration. That is, typically, the desired high molecular weight polysaccharide can be fractionated by a method such as subjecting the polysaccharide to column chromatography having a separation ability in the molecular weight range of 5 to 20 million and fractionating the eluted fraction. Usable gel filtration column products include, for example, the "OHpak SB-800 HQ" series (Showa Denko), the "SUGAR KS-800" series (Showa Denko), the "OHpak SB-2000" series (Showa Denko), the "SUGAR KS-2000" series (Showa Denko), the "Sephacryl" series (Cytiva), and the "Superose" series (Cytiva).

[0042] The obtained preparation may be further subjected to treatments such as dialysis, desalting, ion removal, enzyme treatment, chemical modification, etc., if necessary. In addition, it may be concentrated by evaporation or an acylyzer, or lyophilized.

[0043] The preparation prepared as described above contains a target polysaccharide derived from the cell wall of lactic acid bacteria. The "target polysaccharide" is not limited here, but preferably has a sugar composition ratio in the following range as measured by a known measurement method, for example, the method described in J. Biochemistry (1990), 108 (4), p568-571. Glucose: 45-55% Rhamnose: 25-35% Galactose: 10-20% N-acetylglucosamine: 5-15%

[0044] In addition, the molecular weight estimated from a chromatogram obtained by a gel filtration column and differential refractive index detection is preferably 100,000 or more. In this case, the molecular weight estimated from the chromatogram can be a molecular weight estimated based on a calibration curve obtained from the elution times of the peak tops of the chromatogram when standard samples of known molecular weights across a range of different molecular weights are applied to a gel filtration column under the same conditions.

[0045] The content of the target polysaccharide contained in the preparation is not limited, but may be, for example, 0.1% by mass or more and 100% by mass or less in terms of solid content. The lower limit may be 0.1% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, 9% by mass or more, or 10% by mass or more. The upper limit may be 100% by mass or less, 95% by mass or less, 90% by mass or less, 85% by mass or less, 80% by mass or less, 75% by mass or less, 70% by mass or less, 65% by mass or less, 60% by mass or less, 55% by mass or less, or 50% by mass or less. The content of impurities other than the target polysaccharide is not limited, but may be, for example, 0.001% by mass or more and 99.9% by mass or less in terms of solid content. The upper limit may be 99.9% by mass or less, 95% by mass or less, 90% by mass or less, 85% by mass or less, 80% by mass or less, 75% by mass or less, 70% by mass or less, 65% by mass or less, 60% by mass or less, 55% by mass or less, or 50% by mass or less.

[0046] In this case, the content of the target polysaccharide can be obtained, for example, by calculating the sugar concentration from the color development of the target sample based on a calibration curve obtained from the color development of standard substances of known concentrations by the phenol-sulfuric acid method.

[0047] The cell wall polysaccharide-containing preparation provided by the present invention is not particularly limited in the form of its use, such as food, medicine, cosmetic, supplement, animal feed, etc. For example, the form of cosmetics includes milky lotion, cream, cleansing, massage, sunscreen, makeup base, cream foundation, etc. Note that the meaning of cosmetics includes medicines, quasi-drugs, and cosmetics as defined in the Law on Ensuring Quality, Effectiveness, and Safety of Medicines, Medical Devices, etc.

[0048] The content of the cell wall polysaccharide-containing preparation provided by the present invention in the above-mentioned application form may be appropriately determined according to the purpose of use.Typically, for example, in the case of a cosmetic form, the content may be 0.001% by mass or more and 10% by mass or less, 0.001% by mass or more and 5% by mass or less, 0.01% by mass or more and 3% by mass or less, or 0.01% by mass or more and 1% by mass or less, based on the amount of polysaccharide contained.Also, for example, in the case of a pharmaceutical form, the content may be 0.001% by mass or more and 10% by mass or less, 0.001% by mass or more and 5% by mass or less, 0.01% by mass or more and 3% by mass or less, or 0.01% by mass or more and 1% by mass or less, based on the amount of polysaccharide contained. In addition, in the case of a food product, the amount of polysaccharide contained may be 0.001% by mass or more and 10% by mass or less, or may be 0.001% by mass or more and 5% by mass or less, more preferably 0.01% by mass or more and 3% by mass or less, and may be 0.01% by mass or more and 1% by mass or less. EXAMPLES

[0049] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0050] 1. Materials and Methods (1) Test strains <1> YIT 9029 shares Lacticaseibacillus paracasei YIT 9029 strain. Hereinafter, this strain may be referred to as "YIT 9029 strain."

[0051] <2> Construction of Δcps strain The genome of Lacticaseibacillus paracasei YIT 9029 strain contains a region of approximately 11 kb in length known as the cell wall-associated polysaccharide synthesis (cps) gene cluster (hereinafter sometimes referred to as the "cps1 gene cluster") (Appl. Environ. Microbiol. (2008), 74 (15), p4746-4755.). Table 1 summarizes the 10 genes contained in the cps1 gene cluster, the length of the amino acid sequence of the gene product, and the functions predicted from the amino acid sequence.

[0052] Using the YIT 9029 strain as a host, we constructed a strain in which the cell wall polysaccharide synthesis-related genes (cps1 gene cluster) of approximately 11 kb in total length were completely deleted by double crossover. Hereinafter, this strain will be referred to as the "Δcps strain."

[0053] [Table 1]

[0054] <2.1> Preparation of insert fragment From the genome extracted from the YIT 9029 strain, the primers shown in SEQ ID NO: 21 and 22 were used for DNA approximately 1 kb long upstream of the cps1A gene, and the primers shown in SEQ ID NO: 23 and 24 were used for DNA approximately 1 kb long downstream of the cps1J gene, and the respective DNA fragments were amplified by PCR. At this time, primers were designed so that a sequence equivalent to the 18 bases at each end generated when the plasmid vector pYSSE3 described below was linearized was added to one end of the two types of amplification products obtained (SEQ ID NO: 22 and 23), and at the same time, primers designed to add an overlap region of 16 to 23 bases at the end of each sequence were used to the other end of the two types of amplification products obtained (SEQ ID NO: 21 and 24). Next, both sequences were linked by PCR using this overlap region (using the primers shown in SEQ ID NO: 23 and 22), and an insert fragment of approximately 2 k bases in total was obtained. The reliable linkage was confirmed by PCR using the primers shown in SEQ ID NO: 25 and 26, which amplify 250 bp before and after the junction.

[0055] <2.2> Construction and selection of plasmids containing insert fragments pYSSE3 was used for cloning the insert fragment created as a PCR amplification product. pYSSE3 is a plasmid vector for gene cloning that has an erythromycin resistance gene that functions in both E. coli and lactic acid bacteria as a growth selection marker, and a replication region in E. coli. The PCR amplification product was ligated to this pYSSE3, which had been linearized by PCR, by seamless cloning using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.).

[0056] The inserted fragment was confirmed by colony PCR using the primers shown in SEQ ID NOs: 27 and 28 derived from the pYSSE3 vector sequence. In addition, the nucleotide sequence of the inserted fragment was sequenced using the primers shown in SEQ ID NOs: 25 to 28 as sequence primers to confirm that there were no errors in the sequence.

[0057] <2.3> Introduction of plasmids into YYIT 9029 strain and selection of recombinants The stationary phase YIT 9029 strain was inoculated at 5% (v / v) into 20 mL of a liquid medium for lactic acid bacteria culture, "Difco Lactobacilli MRS broth" (Becton, Dickinson and Company) (hereinafter sometimes referred to as "MRS"), and cultured at 37°C for 2 hours. After cooling on ice for 15 minutes, the mixture was centrifuged at 7,000 rpm for 5 minutes, and the cells were washed with 20 mL of 1 mM HEPES (pH 7). After centrifuging again, the cells were washed with 10 mL of 10% (v / v) glycerol. After centrifuging again, the cells were suspended in 200 μL of 10% (v / v) glycerol, transferred to a 1.5 mL tube, and centrifuged at 15,000 rpm for 5 minutes. After suspending in 100 μL of 10% (v / v) glycerol, 1 μL of the prepared plasmid was added to 40 μL of the mixture and mixed by pipetting. The mixture was added to a 2 mm wide cuvette for electroporation, and a pulse was applied at 25 μF, 200 Ω, and 1.5 KV using an electroporation device "Gene Pulser II" (Bio Rad Laboratories, Inc.). The mixture after pulse application was suspended in 1 mL of MRS liquid medium and cultured at 37 ° C for 1 hour, and then 200 μL / plate was applied to the surface of MRS agar medium containing 20 μg / mL erythromycin with a Conlarge stick. After culturing at 37 ° C for 3 days, some of the colonies that emerged were subjected to colony PCR to confirm whether the plasmid was inserted into the genome at the desired site. Colonies in which the plasmid was confirmed to be inserted were picked up and cultured at 37 ° C for 24 hours in MRS liquid medium containing 20 μg / mL erythromycin.

[0058] <2.4> Obtaining gene disruption strains and confirming gene disruption The insertion of the introduced plasmid into the genome was confirmed by PCR using a primer (SEQ ID NO: 29) derived from the downstream sequence of the cps1 gene group deleted in the target gene disruption strain Δcps strain and the primer shown in SEQ ID NO: 28 derived from the pYSSE3 vector sequence, or similarly by PCR using a primer SEQ ID NO: 30 derived from the upstream sequence of the cps1 gene group and the primer shown in SEQ ID NO: 27 derived from the pYSSE3 vector sequence. The confirmed strain was subcultured for two generations in MRS liquid medium not containing erythromycin. Colonies with distorted shapes compared to the wild type strain were selected on the modified Rogosa plate medium inoculated with the culture liquid, and colony PCR was performed using the primers shown in SEQ ID NOs: 29 and 30 to confirm the deletion of the entire cps1 gene group and the absence of any further deletions at the same site.

[0059] [Table 2]

[0060] (2) Cultivation and cleaning of bacteria Stationary phase bacteria cultured in MRS liquid medium for 24 hours were inoculated at 0.1% (v / v) into 10-30 mL of fresh MRS liquid medium and cultured at 37°C for 24 hours. The resulting bacterial liquid was centrifuged at 10,000 x g for 15 minutes and the supernatant was removed. The same amount of pure water as the original culture medium was added to the precipitate and the bacteria were suspended by pipetting. Centrifugation and suspension were repeated once under the same conditions, and the precipitate after further centrifugation was used as washed bacterial cells.

[0061] (3) Preparation of total cell wall polysaccharide solution The preparation of a polysaccharide extract obtained by extracting cell wall polysaccharides from lactic acid bacteria was carried out as follows. First, L-arabinose (FUJIFILM Wako Pure Chemical Industries, Ltd.) was dissolved in 5 mM Tris-maleate buffer (pH 7.0) to a concentration of 1.5 M. The above-mentioned washed cells were suspended in this and treated with a cell wall lysing enzyme. Specifically, 100 μg of "Lysozyme from chicken egg white lyophilized powder" (Sigma-Aldrich Co.) and 10 μL (100 U) of "Mutanolysin (recombinant)" (A&A Biotechnology) were added per 1 mL of the suspension. This was treated at 50°C for more than 2 hours until clarified. After treatment, the mixture was centrifuged at 10,000 × g for 15 minutes, and the supernatant was passed through a 0.22 μm sterilized filter "33 mm Millex-GP" (Merck KGaA) to remove cell debris. The obtained lysate was added to a centrifugal ultrafiltration filter unit "Amicon Ultra-15 10 kDa MWCO" (Merck KGaA) (nominal molecular weight cutoff: 10,000) and centrifuged at 5,000 × g for 30 minutes. 11 mL of sterile water was added to the retentate, and centrifuged at 5,000 × g for 30 minutes, and this was repeated twice. Approximately 1 mL of the obtained retentate was collected, and 9 mL of 5.5 mM Tris-maleate buffer pH 7.0 (sugar-free) was added, followed by treatment with protease and nuclease. Specifically, 20 μg of a protease preparation "Pronase Protease, Streptomyces griseus" (Merck KGaA) and 20 μg of trypsin (Sigma-Aldrich Co.) were added per 1 mL of solution, and the solution was treated at 37 ° C for 24 hours. The reaction solution was treated at 100°C for 10 minutes to inactivate the enzyme, and then 1 μL of "Benzonase Nuclease" (Merck KGaA) was added per 1 mL of solution, followed by treatment at 37°C for 24 hours. The reaction solution was treated at 100°C for 10 minutes to inactivate the enzyme, followed by addition to a centrifugal ultrafiltration filter unit "Amicon Ultra-15 10 kDa MWCO" (Merck KGaA) (nominal molecular weight cutoff: 10,000) and centrifugal filtration at 5,000 × g for 30 minutes.The retentate was added with 11 mL of pure water and centrifuged at 5,000×g for 30 minutes twice. A total of about 1 mL of the retentate was added to a dialysis tube, Spectra / Por Dialysis Membrane MWCO=6-8,000 (Spectrum Medical Industries), and dialyzed at room temperature for 24 hours. The liquid in the tube after dialysis was used as the total cell wall polysaccharide solution.

[0062] (4) Separation and analysis of polysaccharide-containing preparations by gel filtration chromatography The HPLC system "Alliance 2695 Separations Module" (Waters Corporation) was used for separation and analysis under the conditions in Table 3. The obtained chromatograms were analyzed using the analysis application "Empower" (Waters Corporation) or "Empower 3" (Waters Corporation). Pullulan P100, P200, P400, and P800 (Showa Denko K.K.), polyethylene oxide MW 1,000,000 (PEO; Thermo Fisher Scientific), and polyethylene glycol 2,000,000 (PEG; Fujifilm Wako Pure Chemical Corporation) were used as standards. A calibration curve was created from the molecular weight of the standard (as stated by the manufacturer) and the elution time in HPLC, and the molecular weight of the sample peak was estimated.

[0063] [Table 3]

[0064] (5) Fractionation and recovery of cell wall polysaccharides by gel filtration chromatography Gel filtration chromatography was performed under the separation conditions shown in Table 3, and the eluate was collected in a sample tube at 1-minute intervals from a liquid delivery line directly connected to the column outlet.

[0065] 2. Tests and Results [Test Example 1] (Microscopic Observation) The culture medium of the YIT 9029 and Δcps strains in MRS liquid medium was diluted 10-fold with pure water, and 1 μL was applied to a peel-resistant slide "MAS-coated slide glass" (Matsunami Glass Industry Co., Ltd.). After air drying, the specimens were flame-fixed using a Bunsen burner and Gram stained. The specimens were observed in bright field using an optical microscope "Leica DM5500 B" (Leica Biosystems, Inc.).

[0066] As a result, as shown in Figure 1, when the YIT 9029 and Δcps strains were cultured in MRS liquid medium, autoaggregation of bacterial cells was observed more frequently in the Δcps strain than in the YIT 9029 strain.

[0067] [Test Example 2] (Antibody reactivity) The culture solution of the YIT 9029 strain and the Δcps strain in MRS liquid medium was diluted 10-fold with pure water and centrifuged at 10,000×g for 5 minutes. The pellet was suspended in 0.01M phosphate-buffered saline (PBS) and treated at 100°C for 10 minutes. After centrifugation at 10,000×g for 5 minutes, the pellet was suspended in PBS containing 2% (w / v) bovine serum albumin (BSA) and a specific antibody against cell wall polysaccharides diluted 10-fold, and reacted at 37°C for 30 minutes. After centrifugation at 10,000×g for 5 minutes, the pellet was suspended in PBS and centrifuged again at 10,000×g for 5 minutes, after which the pellet was suspended in PBS containing 2% (w / v) BSA and a secondary antibody "Alexa Fluor 555 goat anti-mouse IgM" (Thermo Fisher Scientific, Inc.) diluted 500-fold, and reacted at 37°C for 30 minutes. After centrifugation at 10,000×g for 5 minutes, the pellet was suspended in PBS twice, and 1 μL of the suspension was applied to each square of a 1 cm×1 cm MAS-coated slide glass (Matsunami Glass Industry Co., Ltd.). After air drying, 4.5 μL / square of fluorescent staining mounting medium "VECTASHIELD Mounting Medium for Fluorescence with DAPI H-1200" (Vector Laboratories, Inc.) was dropped, and a cover glass was placed on top. The specimen was observed under phase contrast (×280), bright field (×700), and fluorescence using an optical microscope "Leica DM5500 B" (Leica Biosystems, Inc.), and digital images were obtained using an imaging system "Leica MMAF system" (Leica Biosystems, Inc.). Fluorescence images were captured in monochrome using the fluorescent dye molecule "Alexa Fluor 555" (excitation light 555 nm, emission fluorescence 585 nm), then converted to green for ease of viewing and overlaid with phase contrast or bright field images on the imaging system "Leica MMAF system."

[0068] As a result, as shown in FIG. 2, fluorescence due to the secondary antibody was observed around all of the bacterial cells in the YIT 9029 strain, but was not observed at all in the Δcps strain treated under the same conditions.

[0069] [Test Example 3] (Isolation of target polysaccharides Part 1) Sterile water was added to the total cell wall polysaccharide solution to make it 10 mL, and centrifuged at 5,000 × g for 2 to 5 minutes using a centrifugal ultrafiltration filter unit "Amicon Ultra-15 100 kDa MWCO" (Merck KGaA) (nominal molecular weight cutoff: 100,000) to make it 1 mL or less. 11 mL of pure water was added to the retentate, and centrifuged at 5,000 × g for 2 to 5 minutes was repeated two more times. The obtained retentate was called "100K retentate". In addition, to reduce the volume of the permeate (total of about 30 mL) obtained in each centrifugal filtration step, centrifugal filtration was repeated at 5,000 × g using a centrifugal ultrafiltration filter unit "Amicon Ultra-15 10 kDa MWCO" (nominal molecular weight cutoff: 10,000) until it became about 1 mL, and the retentate was called "100K permeate".

[0070] FIG. 3 shows the results of separation and analysis by gel filtration column chromatography of the total cell wall polysaccharide solution before ultrafiltration, and the 100K retentate and 100K permeate after ultrafiltration.

[0071] As shown in FIG. 3, when the total cell wall polysaccharide solution of the YIT 9029 strain was fractionated using an ultrafiltration filter (hereinafter sometimes referred to as a "100K filter") with a nominal molecular weight cutoff (NMWL) of 100,000 (100K), it was revealed that some of the peaks showing a high molecular weight side passed through the 100K filter (see the chromatogram in the left column in FIG. 3). That is, the 100K retentate showed a peak with a peak top showing an elution time of 17 minutes on the chromatogram, and the 100K effluent showed a peak with a peak top showing an elution time of 16 minutes on the chromatogram. According to the calibration curve using the standard, the elution time of 16 minutes corresponded to the elution time of a molecular weight of about 1.8 million, and the elution time of 17 minutes corresponded to the elution time of a molecular weight of about 1 million.

[0072] On the other hand, in the case of the Δcps strain, a peak with a top in the 17 min range was observed on the chromatogram of the 100K retentate, but no peak with a top in the 16 min range was detected on the chromatogram of the 100K effluent (see the chromatogram in the right column in Figure 3).

[0073] From the above, it was considered that the peaks of substances with different properties overlapped with the high molecular weight peaks observed when the total cell wall polysaccharide solution of the YIT 9029 strain was separated and analyzed by gel filtration column chromatography. Furthermore, since this phenomenon was not observed in the Δcps strain, it was considered that the substance passing through the 100K filter was cell wall polysaccharide formed by the cps1 gene cluster. Furthermore, since the substance passed through the 100K filter, it was speculated that it was a polysaccharide with a linear structure with few branches and the sugar chain length of the branches.

[0074] [Test Example 4] (Isolation of target polysaccharides, Part 2) A test similar to Test Example 3 was conducted, except that the ultrafiltration membrane used in Test Example 3 was changed to an ultrafiltration filter having a nominal molecular weight cutoff (NMWL) of 30,000 (30K) (centrifugal ultrafiltration filter unit "Amicon Ultra-15 30kDa MWCO" (Merck KGaA)) (hereinafter sometimes referred to as "30K filter").

[0075] As a result, as shown in FIG. 4, a peak was observed in the permeate passing through the 30K filter, which was specific to the YIT 9029 strain and whose peak top showed an elution time of about 16 minutes on the chromatogram (see the chromatogram in the left column in FIG. 4).

[0076] From the above, it was revealed that the separation of high molecular weight polysaccharides passing through an ultrafiltration membrane, which is a phenomenon unique to the YIT 9029 strain, was observed even when an ultrafiltration membrane with a nominal molecular weight cut-off (NMWL) of 30,000 (30K) was used.

[0077] [Test Example 5] (Isolation of target polysaccharides, Part 3) As a control sample for high molecular weight substances, a sample was prepared by adding glucose as an internal standard to polyethylene oxide (Thermo Scientific) with a molecular weight of 1 million (hereinafter sometimes referred to as "PEO+Glc") and subjected to the ultrafiltration membrane in the same manner as in Test Example 3.

[0078] As a result, as shown in FIG. 5, a peak was observed in the permeate passing through the 100K filter, which was specific to the YIT 9029 strain and whose peak top showed an elution time of about 16 minutes on the chromatogram (see the chromatogram in the left column in FIG. 5).

[0079] On the other hand, in the sample containing polyethylene oxide with a molecular weight of 1 million, no substances that could pass through the 100K filter were detected at all (see the chromatogram in the right column in Figure 5).

[0080] From the above, it was revealed that the separation of high molecular weight polysaccharides passing through an ultrafiltration membrane, which is a phenomenon unique to the YIT 9029 strain, was observed even when an ultrafiltration membrane with a nominal molecular weight cutoff (NMWL) of 100,000 (100K) was used. Furthermore, since this phenomenon was not observed in samples containing polyethylene oxide with a molecular weight of 1 million, it was made clear that this was not due to unintended leakage of components caused by the quality of the ultrafiltration membrane used, etc.

Claims

1. A method for producing a cell wall polysaccharide-containing preparation, comprising the steps of: preparing a polysaccharide extract by extracting cell wall polysaccharides from lactic acid bacteria; subjecting the polysaccharide extract to an ultrafiltration membrane; and recovering a target polysaccharide having a molecular weight exceeding the nominal molecular cutoff of the ultrafiltration membrane from the liquid that has passed through the ultrafiltration membrane.

2. 2. The method for producing a cell wall polysaccharide-containing preparation according to claim 1, wherein the permeate that has passed through the ultrafiltration membrane is subjected to further molecular weight fractionation to purify and recover the target polysaccharide.

3. 2. A method for producing a cell wall polysaccharide-containing preparation according to claim 1, wherein the permeate that has passed through the ultrafiltration membrane is further subjected to an additional ultrafiltration membrane having a nominal molecular weight cutoff value less than the nominal molecular weight cutoff value of the ultrafiltration membrane, and the target polysaccharide is recovered in the retentate that does not pass through the additional ultrafiltration membrane.

4. 2. The method for producing a cell wall polysaccharide-containing preparation according to claim 1, further comprising subjecting the permeate that has passed through the ultrafiltration membrane to column chromatography to recover the target polysaccharide in the eluted fraction.

5. 2. The method for producing a cell wall polysaccharide-containing preparation according to claim 1, further comprising a step of subjecting the extract from the lactic acid bacteria to a molecular weight fractionation means different from that of the ultrafiltration membrane before subjecting the extract to the ultrafiltration membrane.

6. 2. The method for producing a cell wall polysaccharide-containing preparation according to claim 1, wherein in the step of preparing the polysaccharide extract, a cell wall lytic enzyme is used to extract cell wall polysaccharides from the lactic acid bacteria.

7. A preparation containing a cell wall polysaccharide, the polysaccharide being derived from a lactic acid bacteria cell wall and passing through an ultrafiltration membrane with a nominal molecular weight cutoff of 30,000, and having a molecular weight of 100,000 or more as estimated from a chromatogram obtained by a gel filtration column and differential refractive index detection.

8. 8. The cell wall polysaccharide-containing preparation according to claim 7, wherein the polysaccharide is derived from Lacticaseibacillus paracasei.

9. 8. The cell wall polysaccharide-containing preparation according to claim 7, wherein the polysaccharide is formed by the cps1 gene cluster of Lacticaseibacillus paracasei.

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