Composition for imparting resistance to environmental stress to bacterium

Abstract

Provided is a technology for imparting resistance to environmental stress to a bacterium such as a lactic acid bacterium. This composition for imparting resistance to environmental stress to a bacterium contains a cps1 gene group derived from Lacticaseibacillus paracasei. The composition is suitably used for imparting resistance to environmental stress caused by at least one selected from the group consisting of acids, bile components, alcohols, metal ions, hydrophobic substances, and surfactants.

Description

Composition for conferring resistance to environmental stress to bacteria

[0001] The present invention relates to the use of a gene cluster that forms cell wall polysaccharides in lactic acid bacteria.

[0002] Probiotics, which aim to promote health through the ingestion of live lactic acid bacteria, have been proposed. It has been reported that the effects of probiotics on cold-like symptoms are alleviated (Non-Patent Documents 1 and 2).

[0003] Meanwhile, in recent years, attempts have been made to utilize components derived from microbial cells as functional components for living organisms. For example, Patent Documents 1 and 2 describe that cell wall-derived polysaccharides present in the cell walls of lactic acid bacteria have the function of regulating the production levels of cytokines such as IL6 and IL12 produced by immunoregulatory cells. According to Non-Patent Documents 3 to 6, it is becoming clear that the molecular entities of lactic acid bacterial cells and cell wall polysaccharides derived therefrom that have such functionality are high-molecular-weight polysaccharides with molecular weights exceeding 100,000.

[0004] JP 2003-73286 A JP 2015-129114 A

[0005] Kan Shida, Tadashi Sato, Ryoko Iizuka, Ryotaro Hoshi, Osamu Watanabe, Tomoki Igarashi, Kouji Miyazaki, Masanobu Nanno, and Fumiyasu Ishikawa middle-aged office workers」Eur J Nutr (2017) 56:45-53.Michael Gleeson, Nicolette C. Bishop, Marta Oliveira, and Pedro Tauler Metabolism, 2010, 1-10.Structure of Polysaccharide-Peptidoglycan Complex from the Cell Wall of Lactobacillus casei YIT9018」J. Biochemistry (1990), 108(4), p568-571.S. Matsumoto , T. Hara , T. Hori , K. Mitsuyama , M. Nagaoka , N. Tomiyasu , A. Suzuki , and M. A. M. .Sata “Probiotic Lactobacillus-induced improvement in murine chronic inflammatory bowel disease is associated with the down-regulation of pro-inflammatory cytokines in lamina propria mononuclear cells” Clin. Exp. Immunol. (2005), 140 (3), p417-426.S. Matsumoto, T. Hara, M. Nagaoka, A. Mike, K. Mitsuyama, T. Sako, M. Yamamoto, S. Kado, and T. Takada, “A component of polysaccharide peptidoglycan complex on Lactobacillus induced an improvement of murine model of inflammatory bowel disease and colitis-associated cancer,” Immunology (2008), 128, e170-e180.Emi Yasuda, Masaki Serata, and Tomoyuki Sako, “Suppressive Effect on Activation of Macrophages by Lactobacillus casei. Strain Shirota Genes Determining the Synthesis of Cell Wall-Associated Polysaccharides” Appl. Environ. Microbiol. (2008), 74 (15), p4746-4755.

[0006] One problem with probiotics is that environmental stresses, such as exposure to low pH during growth or consumption, can impair the activity and integrity of lactic acid bacteria.

[0007] An object of the present invention is to provide a technique for imparting resistance to environmental stress to bacteria such as lactic acid bacteria.

[0008] As a result of intensive research to achieve the above-mentioned object, the inventors discovered that the gene cluster involved in the formation of cell wall polysaccharides in lactic acid bacteria has the function of conferring resistance to environmental stress to the bacteria, thereby completing the present invention.

[0009] That is, in a first aspect, the present invention provides a composition for imparting resistance to environmental stress to bacteria, which comprises the cps1 gene cluster derived from Lacticaseibacillus paracasei.

[0010] In the composition according to the first aspect of the present invention, the cps1 gene group may include the cps1A gene, the cps1B gene, the cps1C gene, the cps1D gene, the cps1E gene, the cps1F gene, the cps1G gene, the cps1H gene, the cps1I gene, and the cps1J gene.

[0011] In the composition according to the first aspect of the present invention, the cps1A gene has the polynucleotide sequence shown in SEQ ID NO: 1 or a polynucleotide sequence having 90% or more homology thereto, the cps1B gene has the polynucleotide sequence shown in SEQ ID NO: 2 or a polynucleotide sequence having 90% or more homology thereto, the cps1C gene has the polynucleotide sequence shown in SEQ ID NO: 3 or a polynucleotide sequence having 90% or more homology thereto, the cps1D gene has the polynucleotide sequence shown in SEQ ID NO: 4 or a polynucleotide sequence having 90% or more homology thereto, the cps1E gene has the polynucleotide sequence shown in SEQ ID NO: 5 or a polynucleotide sequence having 90% or more homology thereto, the cps1F gene has the polynucleotide sequence shown in SEQ ID NO: 6 or a polynucleotide sequence having 90% or more homology thereto, and the cps1G gene has the polynucleotide sequence shown in SEQ ID NO: 7 or a polynucleotide sequence having 90% or more homology thereto, The cps1H gene may have the polynucleotide sequence shown in SEQ ID NO: 8 or a polynucleotide sequence having 90% or more homology thereto; the cps1I gene may have the polynucleotide sequence shown in SEQ ID NO: 9 or a polynucleotide sequence having 90% or more homology thereto; and the cps1J gene may have the polynucleotide sequence shown in SEQ ID NO: 10 or a polynucleotide sequence having 90% or more homology thereto.

[0012] In the composition according to the first aspect of the present invention, the cps1A gene encodes the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having 90% or more homology thereto, the cps1B gene encodes the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having 90% or more homology thereto, the cps1C gene encodes the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having 90% or more homology thereto, the cps1D gene encodes the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having 90% or more homology thereto, the cps1E gene encodes the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having 90% or more homology thereto, the cps1F gene encodes the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having 90% or more homology thereto, and the cps1G gene encodes the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having 90% or more homology thereto. The cps1H gene may encode the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having 90% or more homology thereto; the cps1I gene may encode the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having 90% or more homology thereto; and the cps1J gene may encode the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having 90% or more homology thereto.

[0013] In the composition according to the first aspect of the present invention, the cps1 gene cluster may be used in a form incorporated into a vector.

[0014] In the composition according to the first aspect of the present invention, the environmental stress to which resistance should be imparted may be environmental stress caused by at least one substance selected from the group consisting of acids, bile components, alcohols, metal ions, hydrophobic substances, and surfactants.

[0015] On the other hand, in a second aspect, the present invention provides a method for imparting environmental stress resistance to bacteria, which method comprises the step of introducing the cps1 gene cluster into a target bacterium using the above-mentioned composition.

[0016] The method according to the second aspect of the present invention may impart resistance to environmental stress caused by at least one substance selected from the group consisting of acids, bile components, alcohols, metal ions, hydrophobic substances, and surfactants.

[0017] According to the present invention, a composition and method can be provided that can confer resistance to environmental stress to bacteria by using a group of genes involved in the formation of cell wall polysaccharides in lactic acid bacteria.

[0018]

[0023] This is a table showing an example of an image of the YIT 9029 strain and the Δcps strain when Gram-stained bacterial cells were observed under a microscope in Test Example 1. This is a table showing an example of an image of the YIT 9029 strain and the Δcps strain when the antibody reactivity of the bacterial cells was examined under a fluorescence microscope in Test Example 2. This is a table showing the results of separating and analyzing by gel filtration column chromatography the total cell wall polysaccharide solution before ultrafiltration and the retentate and permeate after ultrafiltration in Test Example 3. This is a table showing the results of examining the acid resistance of the YIT 9029 strain and the Δcps strain in Test Example 4, which shows the survival rate for various treatment times when treated with a low-pH buffer solution whose pH was adjusted with hydrochloric acid. This is a table showing the results of examining the acid resistance of the YIT 9029 strain and the Δcps strain in Test Example 5, which shows the change in growth rate over time when cultured in a pH-unadjusted medium or a low-pH medium (pH 4.5) containing added hydrochloric acid. 1 is a graph showing the results of investigating the acid tolerance of the YIT 9029 strain and the Δcps strain in Test Example 5, and is a graph showing the change in growth fold over time when cultured in a pH-unadjusted medium or a low-pH medium (pH 4.7) supplemented with lactic acid. FIG. 2 is a graph showing the results of investigating the acid tolerance of the YIT 9029 strain and the Δcps strain in Test Example 5, and is a graph showing the growth fold results after 12 hours when cultured in a pH-unadjusted medium or a low-pH medium (pH 4.7) supplemented with lactic acid. FIG. 3 is a graph showing the results of investigating the acid tolerance of the YIT 9029 strain and the Δcps strain in Test Example 5, and is a graph showing the growth fold results after 12 hours when cultured in a pH-unadjusted medium or a low-pH medium (pH 4.7) supplemented with acetic acid. 1 is a graph showing the results of investigating the bile resistance of the YIT 9029 strain and the Δcps strain in Test Example 6, showing the survival rate results when treated for 5 minutes with PBS (pH 7.0) containing deoxycholic acid at a concentration of 0.1% (w / v). 2 is a graph showing the results of investigating the bile resistance of the YIT 9029 strain and the Δcps strain in Test Example 6, showing the survival rate results for various concentrations when treated for 60 minutes with PBS (pH 7.0) containing bovine bile powder.

[0033] This is a table showing the results of investigating the bile tolerance of the YIT 9029 strain and the Δcps strain in Test Example 6, showing the survival rate results for various concentrations and treatment times when treated with PBS (pH 7.0) containing bovine bile powder.

[0034] This is a table showing the results of investigating the acid / bile tolerance of the YIT 9029 strain and the Δcps strain in Test Example 7, showing the survival rate results when treated with a low pH buffer solution (pH 3.0) prepared with hydrochloric acid for 60 minutes, or when further treated with PBS (pH 7.0) containing bovine bile powder at a concentration of 0.2% (w / v).

[0035] This is a table showing the results of investigating the alcohol tolerance of the YIT 9029 strain and the Δcps strain in Test Example 8.

[0036] This is a table showing the results of investigating the metal ion tolerance of the YIT 9029 strain and the Δcps strain in Test Example 9. 1 is a table showing the results of investigating the resistance of the YIT 9029 strain and the Δcps strain to surfactants in Test Example 10.

[0033] FIG. 1 is a table showing the results of investigating the adsorption of the YIT 9029 strain and the Δcps strain to hexadecane in Test Example 11, where the bacterial cell culture solution was treated with hexadecane, the aqueous layer was recovered, and the turbidity (OD615) was measured.

[0034] FIG. 1 is a table showing the results of investigating the adsorption of the YIT 9029 strain and the Δcps strain to hexadecane in Test Example 11, where the bacterial cell culture solution was treated with hexadecane, the aqueous layer was recovered, and the number of bacteria contained in the aqueous layer was measured.

[0035] FIG. 1 is a table showing the results of investigating the zeta potential of the YIT 9029 strain and the Δcps strain in Test Example 12.

[0019] The composition of the present invention is intended to confer resistance to environmental stress to bacteria.

[0020] Here, "resistance to environmental stress" refers to the ability of bacteria to withstand environmental stress without a decrease in biological activity related to viability, proliferation, gene expression, protein expression, enzymatic activity, etc., when exposed to environmental stress, or to minimize any decrease in biological activity. "Conferring" refers to the endowment of bacteria with such an ability, and more specifically, refers to the improvement of the above-mentioned biological activity of bacteria when treated with a composition of the present invention compared to when not treated. In one embodiment, whether or not the composition of the present invention has conferred resistance to environmental stress on bacteria can be evaluated by subjecting bacteria treated with a composition of the present invention and untreated control bacteria to a predetermined stress environment and testing the biological activity of the bacteria.

[0021] Examples of environmental stresses to which bacteria can be made resistant by the composition of the present invention include digestive fluids such as gastric acid and bile, metabolites produced during bacterial fermentation, bactericides, bacteriostatic agents, etc. Examples of material entities that cause environmental stress for bacteria include acids, bile components, alcohols, metal ions, hydrophobic substances, surfactants, etc.

[0022] The acids that cause environmental stress referred to here include, but are not limited to, inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, boric acid, and phosphoric acid, and organic acids such as lactic acid, acetic acid, formic acid, citric acid, and oxalic acid.

[0023] Furthermore, bile components that cause environmental stress include, but are not limited to, cholic acid, glycocholic acid, taurocholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, bile, and the like.

[0024] Furthermore, alcohols that cause environmental stress include, but are not limited to, lower alcohols such as methanol, ethanol, 1-propanol, isopropanol (2-propanol), 1-butanol, 2-butanol, and glycerol, as well as higher alcohols having 6 or more carbon atoms.

[0025] Furthermore, metal ions that cause environmental stress include, but are not limited to, copper ions, silver ions, zinc ions, cobalt ions, nickel ions, and the like.

[0026] Furthermore, hydrophobic substances that cause environmental stress are not limited to, but include, for example, hexane, octane, decane, dodecane, tetradecane, hexadecane, benzene, phenol, toluene, benzoic acid, nitrobenzene, styrene, xylene, cresol, hydroquinone, acetone, chloroform, diethyl ether, etc. As the hydrophobic substance, those with low polarity are more preferable.

[0027] Furthermore, surfactants that cause environmental stress include, but are not limited to, hexadecyltrimethylammonium bromide (CTAB), benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, sodium lauryl sulfate (SDS), alkyldiaminoethylglycine hydrochloride, bile acids, fatty acids, etc. Cationic surfactants are more preferred.

[0028] The composition of the present invention contains the cps1 gene cluster derived from Lacticaseibacillus paracasei. This composition is then introduced into a target bacterium to confer environmental stress resistance to the bacterium. Therefore, from another perspective, the present invention provides a method for conferring environmental stress resistance to a bacterium, comprising the step of introducing the cps1 gene cluster into a target bacterium using the composition. More specifically, the cps1 gene cluster is a gene cluster comprising the cps1A gene, the cps1B gene, the cps1C gene, the cps1D gene, the cps1E gene, the cps1F gene, the cps1G gene, the cps1H gene, the cps1I gene, and the cps1J gene. The DNA sequence of the cps1 gene cluster has been registered in GenBank of the National Center for Biotechnology Information (NCBI) under accession number AB470649.1 and is available to those skilled in the art. Hereinafter, the above gene cluster used in the present invention may be simply referred to as the "cps1 gene cluster."

[0029] Here, the cps1A gene may have the polynucleotide sequence shown in SEQ ID NO: 1 or a polynucleotide sequence having 90% or more homology thereto. It may also encode the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having 90% or more homology thereto. The homology may be 90% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc. This is because a certain level of homology or more allows the functionality of the cps1A gene to be expressed.

[0030] The cps1B gene may have the polynucleotide sequence shown in SEQ ID NO: 2 or a polynucleotide sequence having 90% or more homology thereto. It may also encode the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having 90% or more homology thereto. The homology may be 90% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc. This is because a certain level of homology allows the functionality of the cps1B gene to be expressed.

[0031] Furthermore, the cps1C gene may have the polynucleotide sequence shown in SEQ ID NO: 3 or a polynucleotide sequence having 90% or more homology thereto. It may also encode the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having 90% or more homology thereto. The homology may be 90% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc. This is because a certain level of homology or more allows the functionality of the cps1C gene to be expressed.

[0032] Furthermore, the cps1D gene may have the polynucleotide sequence shown in SEQ ID NO: 4 or a polynucleotide sequence having 90% or more homology thereto. It may also encode the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having 90% or more homology thereto. The homology may be 90% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc. This is because a certain level of homology or more allows the functionality of the cps1D gene to be expressed.

[0033] The cps1E gene may have the polynucleotide sequence shown in SEQ ID NO: 5 or a polynucleotide sequence having 90% or more homology thereto. It may also encode the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having 90% or more homology thereto. The homology may be 90% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc. This is because a certain level of homology allows the functionality of the cps1E gene to be expressed.

[0034] Furthermore, the cps1F gene may have the polynucleotide sequence shown in SEQ ID NO: 6 or a polynucleotide sequence having 90% or more homology thereto. It may also encode the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having 90% or more homology thereto. The homology may be 90% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc. This is because a certain level of homology or more allows the functionality of the cps1F gene to be expressed.

[0035] Furthermore, the cps1G gene may have the polynucleotide sequence shown in SEQ ID NO: 7 or a polynucleotide sequence having 90% or more homology thereto. It may also encode the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having 90% or more homology thereto. The homology may be 90% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc. This is because a certain level of homology or more allows the functionality of the cps1G gene to be expressed.

[0036] Furthermore, the cps1H gene may have the polynucleotide sequence shown in SEQ ID NO: 8 or a polynucleotide sequence having 90% or more homology thereto. It may also encode the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having 90% or more homology thereto. The homology may be 90% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc. This is because a certain level of homology or more allows the functionality of the cps1H gene to be expressed.

[0037] Furthermore, the cps1I gene may have the polynucleotide sequence shown in SEQ ID NO: 9 or a polynucleotide sequence having 90% or more homology thereto. It may also encode the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having 90% or more homology thereto. The homology may be 90% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc. This is because a certain level of homology or more allows the functionality of the cps1I gene to be expressed.

[0038] Furthermore, the cps1J gene may have the polynucleotide sequence shown in SEQ ID NO: 10 or a polynucleotide sequence having 90% or more homology thereto. It may also encode the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having 90% or more homology thereto. The homology may be 90% or more, 96% or more, 97% or more, 98% or more, 99% or more, etc. This is because a certain level of homology or more allows the functionality of the cps1J gene to be expressed.

[0039] For the cps1 gene group and the cps1A gene, cps1B gene, cps1C gene, cps1D gene, cps1E gene, cps1F gene, cps1G gene, cps1H gene, cps1I gene, and cps1J gene contained therein, DNA comprising the polynucleotide sequence can be synthesized using means well known to those skilled in the art, such as PCR or DNA synthesis. Furthermore, cloning, amplification, sequence confirmation, and the like can be performed using conventional cloning vectors. Examples of cloning vectors include pUC18, pUC19, pBluescript II, T-Vector pMD20, and pYSSE3.

[0040] The above-mentioned cps1 gene group and the cps1A gene, cps1B gene, cps1C gene, cps1D gene, cps1E gene, cps1F gene, cps1G gene, cps1H gene, cps1I gene, and cps1J gene contained therein can be introduced into bacterial cells by gene transfer methods well known to those skilled in the art. Examples include chemical transformation, electroporation, viral vectors, and Crispr / Cas9. In one embodiment, although not limited thereto, vectors for introducing foreign genes suitable for specific bacteria are known. DNA containing the gene sequence may be inserted into such vectors, and the vector may then be introduced into bacterial cells. Introduction into bacterial cells can be performed, for example, by electroporation. Examples of vectors for introducing foreign genes include, but are not limited to, pLP10, pYAP300, and pYAP310.

[0041] The composition of the present invention is not particularly limited in form, as long as it contains the above-mentioned cps1 gene cluster or the cps1A gene, cps1B gene, cps1C gene, cps1D gene, cps1E gene, cps1F gene, cps1G gene, cps1H gene, cps1I gene, and cps1J gene contained therein, for example, in the form of a vector incorporated into a vector for introducing a foreign gene as described above, and the form of the composition itself may be, for example, a solid, a liquid, or another form.

[0042] In the present invention, the bacteria to which environmental stress tolerance is imparted are not particularly limited, and examples thereof include lactic acid bacteria. Lactic acid bacteria are not particularly limited, and examples thereof include Lacticaseibacillus bacteria such as Lacticaseibacillus paracasei and Lacticaseibacillus casei, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus cremoris, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus delbrueckii subsp. bulgaricus. Lactobacillus bacteria such as Lactobacillus delbrueckii subsp. 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, and Lactococcus lactis subsp. lactis (Lactococcus lactis subsp. lactis), Lactococcus lactis subsp. cremoris (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 suitable Bifidobacterium species include Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis, and Bifidobacterium globosum. In this specification, the term "lactic acid bacteria" also includes the type of bacteria commonly referred to as bifidobacteria. Lactic acid bacteria may be used singly or in combination of two or more species.

[0043] In recent years, lactic acid bacteria of the genus Lactobacillus have been reclassified. That is, lactic acid bacteria that previously belonged to the genus Lactobacillus have been subdivided into smaller genera, and the genus names of some of the species have been changed.

[0044] (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.

[0045] Therefore, in this specification, they will be indicated by the new classification after the reclassification. Furthermore, for example, among 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 the present application.

[0046] For bacteria into which the above-mentioned cps1 gene group and the cps1A gene, cps1B gene, cps1C gene, cps1D gene, cps1E gene, cps1F gene, cps1G gene, cps1H gene, cps1I gene, and cps1J gene contained therein have been introduced, appropriate media, culture methods, storage methods, etc., which are known depending on the type of bacterium, and therefore, bacteria into which genes have been introduced (transformants) can be grown and maintained according to such known methods suitable for the bacterium. For example, preferred culture media for the lactic acid bacterium 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.), and "MRS broth" (Merck KGaA). The culture may be inoculated into a liquid medium and cultured by stirring or static culture at 30 to 42°C, or may be spread on a plate medium and cultured statically under similar temperature conditions. After culture in a liquid medium, the bacterial cells can be collected by removing the culture supernatant by centrifugation or the like.Alternatively, the collected cells can be suspended in fresh water or an aqueous buffer solution, and then the supernatant removed by centrifugation or the like, thereby washing the cells.

[0047] The bacteria (transformants) into which genes have been introduced according to the present invention can be used as food and drink products, pharmaceuticals, supplements, etc., together with various food ingredients and formulation materials, depending on various desired needs. Preparation into such forms can be carried out by known means for producing food and drink products, or known means for formulating pharmaceuticals or supplements.

[0048] The present invention will be explained in more detail below by way of examples, but these examples are not intended to limit the scope of the present invention.

[0049] [1. Materials and Methods] (1) Test Strains <1> YIT 9029 Strain Lacticaseibacillus paracasei YIT 9029 strain. It has been internationally deposited as Lactobacillus casei YIT 9029 (FERM BP-1366, date of deposit: May 1, 1981) with the International Patent Organism Depositary of the National Institute of Technology and Evaluation (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), the international depositary authority under the Budapest Treaty. Hereinafter, this strain will sometimes be referred to as "YIT 9029 strain."

[0050] <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), pp. 4746-4755.). 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.

[0051] Using the YIT 9029 strain as a host, a strain was constructed in which the cell wall polysaccharide synthesis-related gene cluster (cps1 gene cluster) of approximately 11 kb in total length was completely deleted by double crossover. Hereinafter, this strain will sometimes be referred to as the "Δcps strain."

[0052]

[0053] <2.1> Preparation of Insert Fragments From the genome extracted from the YIT 9029 strain, DNA fragments approximately 1 kb in length upstream of the cps1A gene were amplified by PCR using primers shown in SEQ ID NOs: 21 and 22, and DNA fragments approximately 1 kb in length downstream of the cps1J gene were amplified using primers shown in SEQ ID NOs: 23 and 24. Primers (SEQ ID NOs: 22 and 23) were designed to add to one end of each of the two resulting amplification products a sequence equivalent to the 18 base pairs generated upon linearization of the plasmid vector pYSSE3 (described below). Primers (SEQ ID NOs: 21 and 24) were also designed to add to the other end of each of the two resulting amplification products a 16- to 23-base overlap region. Next, these two sequences were linked by PCR using the overlap region (primers shown in SEQ ID NOs: 23 and 22), yielding an insert fragment totaling approximately 2 kb in length. The complete ligation was confirmed by PCR using primers shown in SEQ ID NOs: 25 and 26, which amplify 250 bp before and after the ligation site.

[0054] <2.2> Preparation and selection of plasmids containing insert fragments pYSSE3 was used for cloning the insert fragments created as PCR amplification products. pYSSE3 is a plasmid vector for gene cloning that contains 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 linearized by PCR by seamless cloning using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.).

[0055] The inserted fragment was confirmed by colony PCR using the primers derived from the pYSSE3 vector sequence shown in SEQ ID NOs: 27 and 28. The nucleotide sequence of the inserted fragment was also 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.

[0056] <2.3> Introduction of Plasmid into YIT 9029 Strain and Selection of Recombinants Stationary-phase YIT 9029 strain was inoculated at 5% (v / v) into 20 mL of 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 15 minutes of ice cooling, the culture 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 the mixture 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 electroporation cuvette, 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.). After applying the pulse, the mixture was suspended in 1 mL of MRS liquid medium and cultured at 37°C for 1 hour. 200 μL / plate was then applied to the surface of MRS agar medium containing 20 μg / mL erythromycin using a Conlarge stick. After culturing at 37°C for 3 days, some of the resulting colonies were subjected to colony PCR to confirm insertion into the genome at the target site. Colonies in which plasmid insertion was confirmed were picked and cultured at 37°C for 24 hours in MRS liquid medium containing 20 μg / mL erythromycin.

[0057] <2.4> Obtaining a Gene-Disrupted Strain and Confirming Gene Disruption The integration of the introduced plasmid into the genome was confirmed by PCR using a primer (SEQ ID NO: 29) derived from the sequence downstream of the cps1 gene cluster deleted in the target gene-disrupted strain, Δcps, and the primer shown in SEQ ID NO: 28 derived from the pYSSE3 vector sequence, or similarly by PCR using primer SEQ ID NO: 30 derived from the sequence upstream of the cps1 gene cluster and the primer shown in SEQ ID NO: 27 derived from the pYSSE3 vector sequence. The confirmed strains were passaged for two generations in erythromycin-free MRS liquid medium. Colonies with distorted shapes compared to the wild-type strain were selected on modified Rogosa plates inoculated with the culture medium, 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 cluster and the absence of further deletions at the same site.

[0058]

[0059] (2) Cultivation and washing of bacteria Stationary-phase bacteria cultured in MRS liquid medium for 24 hours were inoculated at 0.1% (v / v) into 10 to 30 mL of fresh MRS liquid medium and cultured at 37°C for 24 hours. The resulting bacterial solution was centrifuged at 10,000 × g for 15 minutes, and the supernatant was removed. An equal volume of pure water to 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.

[0060] (3) Preparation of Total Cell Wall Polysaccharide Solution A polysaccharide extract solution was prepared by extracting cell wall polysaccharides from lactic acid bacteria 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 washed bacterial cells were suspended in this solution and treated with a cell wall-lytic 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 mL of suspension. This solution was treated at 50°C for at least 2 hours until clarified. After treatment, the solution was centrifuged at 10,000 × g for 15 minutes, and the supernatant was passed through a 0.22 μm sterile filter "33 mm Millex-GP" (Merck KGaA) to remove bacterial debris. The resulting 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 this process was repeated twice. Approximately 1 mL of the resulting retentate was collected, and 9 mL of 5.5 mM Tris-maleate buffer, pH 7.0 (sugar-free) was added. The resulting solution was then treated with protease and nuclease. Specifically, 20 μg of the protease preparation "Pronase Protease, Streptomyces griseus" (Merck KGaA) and 20 μg of trypsin (Sigma-Aldrich Co.) were added per mL of solution, and the mixture 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 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, and then the reaction solution 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.The retentate was added with 11 mL of purified water and centrifuged at 5,000 × g for 30 minutes, and this process was repeated twice. Approximately 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 solution in the tube after dialysis was used as the total cell wall polysaccharide solution.

[0061] (4) Separation and Analysis of Polysaccharide-Containing Preparations by Gel Filtration Chromatography Separation and analysis were performed using an HPLC system "Alliance 2695 Separations Module" (Waters Corporation) under the conditions listed in Table 3. The resulting chromatograms were analyzed using the analytical 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 MW 2,000,000 (PEG; Fujifilm Wako Pure Chemical Corporation) were used as standards. A calibration curve was created based on the molecular weights of the standards (as specified by the manufacturer) and their HPLC elution times, and the molecular weights of the sample peaks were estimated.

[0062]

[0063] 2. Tests and Results Test Example 1 (Microscopic Observation) Culture solutions of the YIT 9029 strain and the Δcps strain in MRS liquid medium were diluted 10-fold with pure water, and 1 μL of the solution was applied to a peel-resistant slide, "MAS-Coated Slide Glass" (Matsunami Glass Industry Co., Ltd.). After air-drying, the sample was flame-fixed using a Bunsen burner and then Gram stained. The sample was observed under bright field conditions using an optical microscope, "Leica DM5500 B" (Leica Biosystems, Inc.).

[0064] As a result, as shown in Figure 1, when microscopic images of the YIT 9029 strain and the Δcps strain cultured in MRS liquid medium were observed, autoaggregation of bacterial cells was observed more frequently in the Δcps strain than in the YIT 9029 strain.

[0065] Test Example 2 (Antibody Reactivity) Culture solutions of YIT 9029 strain and Δcps strain in MRS liquid medium were diluted 10-fold with pure water and centrifuged at 10,000 × g for 5 minutes. The pellet was suspended in 0.01 M phosphate-buffered saline (PBS) and incubated 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 10-fold diluted antibody specific to cell wall polysaccharides, and incubated 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. The pellet was then suspended in PBS containing 2% (w / v) BSA and a 500-fold diluted secondary antibody, "Alexa Fluor 555 goat anti-mouse IgM" (Thermo Fisher Scientific, Inc.), and incubated 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 well of a 1 cm × 1 cm MAS-coated slide (Matsunami Glass Industry Co., Ltd.). After air-drying, 4.5 μL of fluorescent staining mounting medium, "VECTASHIELD Mounting Medium for Fluorescence with DAPI H-1200" (Vector Laboratories, Inc.), was added per well and a cover glass was placed on top. The specimens were observed using a light microscope "Leica DM5500 B" (Leica Biosystems, Inc.) under phase contrast (×280), bright-field (×700), and fluorescence conditions, and digital images were acquired 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. The images were then overlaid with the phase contrast or bright-field images on the imaging system "Leica MMAF system."

[0066] As a result, as shown in FIG. 2, fluorescence due to the secondary antibody was observed around all of the cells in the YIT 9029 strain, but was not observed at all in the Δcps strain treated under the same conditions.

[0067] Test Example 3 (Separation and Analysis of Total Cell Wall Polysaccharide Solution) Sterile water was added to the total cell wall polysaccharide solution to make 10 mL, and the solution was 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 reduce the volume to less than 1 mL. 11 mL of purified water was added to the retentate, and this process was repeated twice more, this time centrifuging at 5,000 × g for 2 to 5 minutes. The resulting retentate was designated "100K retentate." Furthermore, to reduce the volume of the permeate (approximately 30 mL in total) 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) to make the volume approximately 1 mL, and the resulting retentate was designated "100K permeate."

[0068] 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.

[0069] As shown in Figure 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 found that a portion of the high-molecular-weight peak passed through the 100K filter (see the chromatogram in the left column of Figure 3). Specifically, the 100K retentate showed a peak with an elution time of approximately 17 minutes on the chromatogram, while the 100K effluent showed a peak with an elution time of approximately 16 minutes on the chromatogram. According to a calibration curve using a standard, the elution time of 16 minutes corresponded to the elution time of a molecule with a molecular weight of approximately 1.8 million, and the elution time of 17 minutes corresponded to the elution time of a molecule with a molecular weight of approximately 1 million.

[0070] On the other hand, in the case of the Δcps strain, a peak with a top in the 17-minute range was observed on the chromatogram of the 100K retentate, but no peak with a top in the 16-minute range was detected on the chromatogram of the 100K effluent (see the chromatogram in the right column in Figure 3).

[0071] Based on the above, it was suggested that the high molecular weight peak observed when the total cell wall polysaccharide solution of the YIT 9029 strain was separated and analyzed by gel filtration column chromatography overlapped with peaks of substances with different properties. Furthermore, since this phenomenon was not observed in the Δcps strain, it was suggested 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 to be a polysaccharide with a linear structure, with few branches and few sugar chains.

[0072] Test Example 4 (Evaluation of Acid Tolerance, Part 1) The Δcps strain, which is unable to form normal cell wall polysaccharides due to the deletion of the cps1 gene cluster, was evaluated for its acid resistance using the parent strain, YIT 9029, which has normal cell wall polysaccharides, as a control.

[0073] Specifically, the test was performed as follows. Culture solutions of the YIT 9029 strain or the Δcps strain in MRS liquid medium were prepared, and 90.9 μL of each solution was added to 9 mL of 0.2 M glycine-HCl buffer (37°C) adjusted to various pH values. The mixture was mixed using a vortex mixer and then allowed to stand at 37°C. After various predetermined times had elapsed, 1 mL of the treatment solution was collected and added to 0.01 M PBS (pH 7.2-7.4). The appropriate samples were then diluted with PBS and plated on MRS plates. The agar plates on which the samples had been plated were cultured at 37°C under aerobic conditions for at least two days. The number of colonies that emerged was counted, and the number of colony-forming units (CFU) per mL of bacterial solution was calculated from the dilution factor. This CFU count was used as the viable cell count, and the survival rate was calculated from the viable cell count in the culture solution before treatment and the viable cell count determined after each treatment time. It was confirmed that the survival rates of both the YIT 9029 strain and the Δcps strain did not decrease for at least four hours after the start of treatment with a 0.2 M glycine aqueous solution (pH 6.2) without pH adjustment (almost 100%). Furthermore, the pH fluctuations after the start of treatment for all treatment solutions adjusted to various pHs were less than 0.1.

[0074] FIG. 4 shows the average survival rate results from five independent tests, along with their standard deviations.

[0075] As shown in Figure 4, when the bacterial cells were treated with a glycine-HCl buffer solution adjusted to pH 2.6-3.0 with hydrochloric acid, the survival rate of the Δcps strain was significantly reduced compared to that of the YIT 9029 strain. In particular, when treated at pH 2.8 and pH 3.0, the viable cell count of the Δcps strain was reduced by 30% or more, even when the reduction in viable cell count of the YIT 9029 strain was less than 1% (60 minutes after the start of treatment).

[0076] Test Example 5 (Evaluation of Acid Tolerance, Part 2) The Δcps strain, which is unable to form normal cell wall polysaccharides due to the deletion of the cps1 gene cluster, was evaluated for its growth ability under low pH conditions using the parent strain YIT 9029, which has normal cell wall polysaccharides, as a control.

[0077] Specifically, the test was carried out as follows: MRS liquid medium, the pH of which had been adjusted to 4.5 to 4.7 with hydrochloric acid, lactic acid, or acetic acid, or MRS without pH adjustment (pH 6.2 to 6.3), was inoculated with the YIT 9029 strain or Δcps strain in the stationary phase, which had been separately cultured in MRS liquid medium for 24 hours, and cultured at 37°C for 3 to 24 hours. After the lapse of various predetermined times, a portion of the culture medium was sampled, and the viable cell count was measured in the same manner as in Test Example 4. The growth fold was calculated using the inoculated viable cell count as the denominator.

[0078] The results are shown in Figures 5 to 8.

[0079] Figure 5 shows the results of culturing in a low pH medium containing hydrochloric acid, where the growth curves are averaged over five independent culture tests and their standard deviations are shown.

[0080] As shown in Figure 5, no difference in growth fold was observed between the YIT 9029 strain and the Δcps strain in unadjusted pH medium (pH 6.3). In contrast, in medium adjusted to pH 4.5 with the addition of hydrochloric acid, the growth fold of the Δcps strain was significantly lower at all measurement times after 3 hours from the start of culture. The growth fold ratio between the strains (YIT 9029 strain / Δcps strain) reached a maximum of 2.2 at 12 hours after the start of culture and was 1.4 at 24 hours.

[0081] Figures 6 and 7 show the results of culturing in a low pH medium supplemented with lactic acid. Figure 7 shows the average results of five independent culturing experiments, along with the standard deviation.

[0082] As shown in Figure 6, when the growth curves were examined (n = 1), no clear difference in the growth fold between the YIT 9029 strain and the Δcps strain was observed in the unadjusted medium (pH 6.3). In contrast, in the medium adjusted to pH 4.7 with the addition of lactic acid, the growth fold of the Δcps strain was lower at all measurement times after 3 hours from the start of culture. The growth fold ratio between the strains (YIT 9029 strain / Δcps strain) reached a maximum of 2.7 at 15 hours after the start of culture and decreased to 1.7 at 24 hours.

[0083] 7 , when growth was compared after 12 hours of culture (n=5), there was almost no difference in growth fold between the YIT 9029 strain and the Δcps strain in a medium without pH adjustment (pH 6.3), with the growth fold ratio between the strains (YIT 9029 strain / Δcps strain) being only 1.2. In contrast, in a medium adjusted to pH 4.7 with the addition of lactic acid, the growth fold ratio between the strains (YIT 9029 strain / Δcps strain) was 2.2, a result similar to that obtained when hydrochloric acid was added to the medium in Test Example 4.

[0084] Figure 8 shows the results of culturing in a low pH medium containing acetic acid, where the average results of five independent culturing experiments are shown together with the standard deviation.

[0085] As shown in Fig. 8, when growth was compared after 12 hours of culture (n = 5), no significant difference in growth fold was observed between the YIT 9029 strain and the Δcps strain in a medium without pH adjustment (pH 6.2). In contrast, in a medium adjusted to pH 4.7 with the addition of acetic acid, the growth fold of the Δcps strain was significantly lower, with the growth fold ratio between the strains (YIT 9029 strain / Δcps strain) being 1.6.

[0086] Test Example 6 Evaluation of Bile Resistance The Δcps strain, which is unable to form normal cell wall polysaccharides due to the deletion of the cps1 gene cluster, was evaluated for bile resistance using the parent strain YIT 9029, which has normal cell wall polysaccharides, as a control.

[0087] Specifically, the test was carried out as follows: First, a predetermined amount of sodium deoxycholate (Sigma Aldrich) or bovine bile powder (Difco Oxgall, Becton, Dickinson and Company) was added to phosphate-buffered saline (pH approximately 7.3) and sterilized with a 0.22 μm filter.

[0088] The component concentrations and pH of each treatment solution are as follows: Deoxycholic acid: 0.1% (w / v), pH 7.0 Bovine bile (equivalent to 10% (w / v) powder): 0.02 to 0.8% (w / v), pH 7.0

[0089] Separately, culture solutions of the YIT 9029 strain or the Δcps strain in MRS liquid medium were prepared, and 100 μL of each was added to 9.9 mL of each treatment solution kept at 37° C., mixed with a vortex mixer, and then allowed to stand at 37° C. After the lapse of various predetermined times, a portion of the treatment solution was sampled, and the viable cell count was measured in the same manner as in Test Example 4. The survival rate was calculated from the viable cell count in the culture solution before treatment and the viable cell count determined after each treatment time had elapsed.

[0090] The results are shown in Figures 9 to 11.

[0091] Figure 9 shows the results of examining the effect of treatment with deoxycholic acid for 5 minutes, showing the average survival rate from five independent tests together with the standard deviation.

[0092] 9, the survival rate of the Δcps strain was significantly lower than that of the YIT 9029 strain. Specifically, the average survival rate of the YIT 9029 strain was about 8 times higher than that of the Δcps strain.

[0093] Figure 10 shows the results of examining the effect of treatment with bovine bile for 60 minutes, showing the average survival rate results from five independent tests together with the standard deviation.

[0094] 10, the survival rate of the Δcps strain tended to be lower than that of the YIT 9029 strain. In particular, it was significantly lower at a bovine bile concentration of 0.2% (w / v).

[0095] FIG. 11 shows the results of a detailed investigation into the time course of survival rates at various concentrations of bovine bile (n=1).

[0096] 11A to 11C, regardless of the bacterial strain or bovine bile concentration, the survival rate decreased rapidly within 5 minutes of the start of treatment, and then gradually decreased until 60 minutes had elapsed. At this time, the survival rate of the Δcps strain was lower than that of the YIT 9029 strain, with a difference of approximately 2- to 6-fold.

[0097] Test Example 7 Evaluation of Acid and Bile Resistance The Δcps strain, which is unable to form normal cell wall polysaccharides due to the deletion of the cps1 gene cluster, was evaluated for its acid and bile resistance using the parent strain YIT 9029, which has normal cell wall polysaccharides, as a control.

[0098] Specifically, the test was conducted as follows. First, a 0.2 M glycine-hydrochloric acid buffer solution with a pH of 3.0 prepared in Test Example 4, or a phosphate-buffered saline solution (pH 7.0) with a bovine bile concentration of 0.2% (w / v) prepared in Test Example 6, was prepared. Separately, a culture solution of the YIT 9029 strain or the Δcps strain in MRS liquid medium was prepared, and 1 mL of each was added to 9 mL of 0.2 M glycine-hydrochloric acid buffer solution (pH 3.0) kept at 37°C, mixed with a vortex mixer, and then allowed to stand at 37°C. After 60 minutes, a portion of the treated solution was collected, and the survival rate of this sample was calculated in the same manner as in Test Example 4. When further treatment with bovine bile was performed, 1 mL of the treatment solution was taken and added to 9 mL of phosphate-buffered saline (pH 7.0) containing 0.2% (w / v) bovine bile that had been kept at 37° C., mixed with a vortex mixer, and then allowed to stand at 37° C. After 60 minutes had elapsed, a portion of the treatment solution was sampled, and the survival rate of this sample was calculated in the same manner as in Test Example 4.

[0099] The results are shown in Figure 12.

[0100] 12, treatment with 0.2 M glycine-HCl buffer (pH 3.0) prepared with hydrochloric acid for 60 minutes reduced the survival rate of only the Δcps strain. Furthermore, after subsequent treatment with 0.2% (w / v) bovine bile for 60 minutes, the survival rate of the YIT 9029 strain was 73%, while that of the Δcps strain decreased to 19%.

[0101] Test Example 8 (Evaluation of Alcohol Tolerance) Treatment of Bacterial Cells with Alcohol The culture solution of the YIT 9029 strain or the Δcps strain (approximately 8 × 10 ８CFU / mL) was added to 9.9 mL of aqueous solutions containing alcohol (methanol, ethanol, isopropanol, or 1-butanol) at various concentrations, and the mixture was allowed to stand at room temperature for 10 minutes. The number of viable bacteria was measured before and after treatment, and the ratio of the number of viable bacteria added as the denominator was used as the survival rate. The average survival rate obtained from three independent tests was calculated. The lower limit of viable bacteria measurement was 200 CFU / mL (survival rate approximately 0.002%). Data below the lower limit of measurement was shown on the graph as a survival rate of 0.001%, but was not included in the calculation of the average value.

[0102] Measurement of viable cell count After diluting the culture solution appropriately with physiological saline, the diluted bacterial solution was applied to a previously prepared MRS plate medium using a spiral plater EDDY JET 2 (IUL Instruments GmbH). After culturing the medium at 37°C for 2 days or more, the colonies grown on the medium were counted using an automatic colony counter Protocol 3 (Synoptics Ltd.), and the resulting CFU number was taken as the viable cell count.

[0103] Results As shown in FIG. 13, in all cases of alcohol, the YIT9029 strain tended to have a higher survival rate than the Δcps strain.

[0104] Test Example 9 (Evaluation of Metal Ion Resistance) Evaluation of Growth Ability in the Presence of Metal Ions Copper oxide fine powder (CuO(II), nanopowder, <50 nm particle size, Sigma) was added to MRS liquid medium at 0.0025% to 0.01% (w / v). MRS cultures of the YIT 9029 strain or the Δcps strain in the stationary phase were inoculated at 0.05% (v / v) or 0.1% (v / v), respectively, and cultured at 37°C (the viable cell count at the start of culture was approximately 1.5 × 10 for both strains). ６ CFU / mL). The viable cell count and pH were measured over time up to 24 hours. The growth rate was calculated assuming the viable cell count at the start of culture was 1, and the average growth rate obtained from five independent tests was calculated.

[0105] Measurement of viable cell count: Measurement was carried out in the same manner as in Test Example 8.

[0106] Results: As shown in Figure 14, when CuO was not added, there was no difference in growth fold until 8 hours of culture. After 15 hours, the YIT 9029 strain showed a significant increase, but the difference was approximately 2-fold. On the other hand, when CuO was added, growth of the Δcps strain was delayed, and significant differences were observed at 15 hours of culture at all concentrations tested. At this time, the growth fold of the YIT 9029 strain was approximately 4-fold, 9-fold, and 23-fold that of the Δcps strain at CuO concentrations of 0.0025% (w / v), 0.005% (w / v), and 0.01% (w / v), respectively. After 24 hours of cultivation, the growth rate of the Δcps strain had caught up to within 2-fold of the YIT 9029 strain, even at CuO concentrations of 0.0025% (w / v) and 0.005% (w / v). However, at a CuO concentration of 0.01%v, there was a significant difference between the YIT 9029 strain and the Δcps strain from 8 hours onwards until 24 hours later, with the growth rate ratio of the YIT 9029 strain being approximately 2-fold, 23-fold, and 6-fold that of the Δcps strain at the respective times.

[0107] From the above, it was revealed that the Δcps strain has weaker resistance to copper ions than the YIT 9029 strain.

[0108] Regarding pH, large differences were observed between the strains at CuO concentrations of 0.005% (w / v) and 0.01% (w / v). At 15 hours of culture, the pH did not reach 4.8, the pH at which the growth of the Δcps strain begins to stagnate in MRS medium. In particular, the average pH of the Δcps strain at 15 hours of culture at a CuO concentration of 0.01% (w / v), where the difference in growth fold was greatest, was relatively high at 5.8. This suggests that the difference in growth fold between the strains is not due to differences in acid tolerance between the strains, but rather to differences in tolerance to the presence of copper ions.

[0109] Separately, microscopic observation of the morphology of strains after culturing in the presence of CuO revealed that deformation (shrinkage and rounding) of almost all cells of the Δcps strain was observed at a CuO concentration of 0.0025% (w / v). On the other hand, no deformed cells were observed in the YIT 9029 strain at a CuO concentration of 0.0025% (w / v), deformation began to be observed in a small number of cells (approximately 10%) at a CuO concentration of 0.005% (w / v), and significant deformation was observed in almost all cells at a CuO concentration of 0.01% (w / v).

[0110] Test Example 10 (Evaluation of Resistance to Surfactants) Evaluation of Growth Ability in the Presence of Surfactants Cationic surfactants, hexadecyltrimethylammonium bromide (CTAB, Sigma) and benzalkonium chloride (Takeda Pharmaceutical Co., Ltd.), were adjusted to concentrations of 0.63 to 80 μM by serial two-fold dilution with pure water. Similarly, anionic surfactant, sodium lauryl sulfate (SDS), was adjusted to concentrations of 0.06 to 8 mM with pure water. Each solution was dispensed at 50 μL per well into a 96-well plate. Approximately 1 × 10 YIT 9029 strain or Δcps strain was added to each well. ６ 50 μL of MRS liquid medium inoculated to a concentration of CFU / mL was added to each well and cultured at 37°C for 24 hours. After culture, the 96-well plate was visually observed from the top and bottom, and the minimum concentration at which no bacterial precipitation occurred for each substance was defined as the minimum inhibitory concentration (MIC). The geometric mean of the MIC values ​​obtained from three independent tests was calculated.

[0111] 15 , in the case of the cationic surfactants CTAB and benzalkonium chloride, the average MIC values ​​for the YIT 9029 strain were 2.0 to 2.5 times higher than that of the Δcps strain, demonstrating that the Δcps strain has weaker resistance to cationic surfactants than the YIT 9029 strain. On the other hand, in the case of the anionic surfactant sodium lauryl sulfate (SDS), the average MIC value for the YIT 9029 strain was 1.3 times higher than that of the Δcps strain, demonstrating a tendency for the Δcps strain to have weaker resistance to anionic surfactants than the YIT 9029 strain.

[0112] Test Example 11 (Adhesion to Hexadecane) - Treatment of Bacterial Cells with Hexadecane Stationary-phase YIT 9029 or Δcps strains cultured in MRS liquid medium for 24 hours were precipitated by centrifugation at 10,000 × g for 15 minutes. The supernatant was removed and suspended in an equal volume of PUM buffer (16.9 g / L KHPO, 7.26 g / L KHPO, 1.8 g / L urea, 0.2 g / L MgSO.7H0), followed by centrifugation and suspension again. Using a PiCOSCOPE PAS-110 spectrophotometer (Ushio Inc.), the suspension was diluted with PUM buffer to a turbidity (OD615) of approximately 0.5 at 615 nm (maximum absorption wavelength; measurement wavelength range: 575-660 nm). 1.2 mL of the suspension was dispensed into a 15 mL sample tube, and 0.2 to 4.8 mL of hexadecane was added from above. After stirring for 30 seconds with a vortex mixer, the tube was left to stand for 30 minutes. After observing the appearance, the aqueous layer was separated and the OD615 and total bacterial count were measured.

[0113] Measurement of total bacterial count The culture solution in MRS liquid medium or the aqueous layer containing bacteria was diluted 50-fold with phosphate-buffered saline, and 2.7 μL of the diluted solution was added to one frame of a 1 cm × 1 cm water-repellent printed glass slide (Matsunami Glass Industry Co., Ltd.). A cover glass was placed over the slide, and photographs were taken of 100 or more fields of view at 400x magnification using a digital microscope VHX-8000 (Keyence Corporation). The fields of view were connected, and the area of ​​the entire frame (100 mm ２ ) 1 / 6 (16.65 mm ２ The cells within the range of 100 μm were automatically extracted and counted.

[0114] Results: Regarding the appearance after the test, the emulsion droplets formed after stirring were visually larger for the Δcps strain. Furthermore, the Δcps strain also showed a greater decrease in turbidity (increase in transparency) in the separated aqueous layer.

[0115] As shown in FIG. 16, a decrease in the OD615 of the aqueous layer was observed with an increase in the ratio of hexadecane for both strains, but a significant decrease was observed for the Δcps strain compared to the YIT 9029 strain.

[0116] As shown in FIG. 17 , when the number of bacteria in the aqueous layer was examined, it was found that, as with OD615, the number of bacteria in the aqueous layer decreased with increasing hexadecane ratio for both strains, but a significant decrease was observed for the Δcps strain compared to the YIT 9029 strain.

[0117] Furthermore, when the destination of the Δcps strain bacterial cells was investigated, they accumulated at high density inside the emulsion droplets (water layer), while almost no bacterial cells were observed in the oil layer.

[0118] As described above, it was revealed that the Δcps strain has a higher affinity for hexadecane than the YIT 9029 strain. This is thought to be because the Δcps strain does not produce cell wall polysaccharides, resulting in increased hydrophobicity of the bacterial cell surface.

[0119] Test Example 12 (Zeta Potential Measurement) A 1% (v / v) preculture of the bacterial strain was inoculated into MRS liquid medium and cultured at 37°C for 24 hours. The mixture was then centrifuged at 10,000 × g for 5 minutes, and the precipitate was washed twice with an equal volume of ultrapure water (Milli-Q) to the MRS liquid medium used for culture. The turbidity at 615 nm (OD615) measured using a PiCOSCOPE PAS-110 spectrophotometer (Ushio Inc.) was adjusted with ultrapure water (Milli-Q) to 1.0, and this was used as a bacterial suspension. Immediately before measuring the zeta potential, 0.2 mL of the bacterial suspension was added to 1.8 mL of 5 mM potassium phosphate buffer (pH 7.0, pH 4.8, or pH 4.0), and 1 mL of this mixture was filled into a capillary cell DTS1070 (Malvern Instruments Ltd.). The cells were treated with ultrasound for 15 seconds, and then measurements were taken three times per sample using a Zetasizer Nano (Malvern Instruments Ltd.) to calculate the average zeta potential.

[0120] As shown in Figure 18, there was no significant difference between the two tests, and the absolute value of the negative zeta potential generally decreased with decreasing pH. The potential of the YIT 9029 strain fluctuated within the range of -0.8 to -4.7 mV, while that of the Δcps strain fluctuated within the range of -4.4 to -14.2 mV with changes in pH. At the same pH, the absolute value of the potential of the Δcps strain was always approximately 3 to 4 times greater than that of the YIT 9029 strain.

[0121] As described above, it was revealed that the Δcps strain exhibited a larger negative shift in zeta potential than the YIT 9029 strain. This is because the Δcps strain does not produce cell wall polysaccharides, and the characteristics of the bacterial cell surface are H ＋ This was thought to be due to an increase in the amount of ion attractants (a decrease in the pH around the bacterial cells).

[0122] Preparation Example 1 (Construction of a Plasmid Vector for Introducing the cps1 Gene Cluster) A plasmid vector incorporating a cell wall polysaccharide synthesis-related gene cluster (cps1 gene cluster) was constructed.

[0123] Specifically, DNA corresponding to a genomic region of approximately 11 kb in length containing the cps1 gene cluster consisting of the above-mentioned 10 genes was obtained by PCR using the genomic DNA of the Lacticaseibacillus paracasei YIT 9029 strain as a template. Table 4 shows the primer sets used. The primer shown in SEQ ID NO: 31 is a primer containing the sequence of a region containing a ribosome binding site sequence upstream of the cps1A gene and a sequence containing a SmaI cleavage site on its 5'-end, and the primer shown in SEQ ID NO: 32 is a primer containing the sequence of a complementary strand of a region downstream of the cps1J gene and a sequence containing a PstI cleavage site on its 5'-end.

[0124]

[0125] PCR was performed using the above primer set and KOD Plus DNA polymerase (Toyobo Co., Ltd.) according to the instructions included with the product. After the reaction, the amplified DNA was purified by phenol-chloroform extraction and ethanol precipitation. This DNA was cleaved with restriction enzymes SmaI and PstI (Takara Bio Inc.) at 37°C for 20 hours, and then again by phenol-chloroform extraction and ethanol precipitation to purify the DNA fragment consisting of the cps1 gene cluster.

[0126] Meanwhile, pYAP300 was prepared as a plasmid vector. This plasmid vector contains the attP site and int gene derived from phage FSW and is designed for site-specific integration into the attB site on the genome of Lacticaseibacillus casei. This plasmid DNA was linearized by cleavage with restriction enzymes SmaI and PstI (Takara Bio Inc.) at 37°C for 20 hours. After the reaction, the linearized plasmid DNA was purified by phenol-chloroform extraction and ethanol precipitation. Then, 20 μL of 10x CIP buffer (Toyobo Co., Ltd.) and water were added to make a volume of 200 μL, and 3 μL of calf intestine phosphatase (CIP) (Toyobo Co., Ltd.) was added and incubated at 37°C for 2 hours to dephosphorylate the termini. After the reaction, the linearized plasmid DNA was purified by phenol-chloroform extraction and ethanol precipitation.

[0127] The DNA fragment comprising the cps1 gene cluster was ligated with pYAP300 as follows. Approximately 0.01 to 0.1 μg of each of the DNA fragment comprising the cps1 gene cluster and linearized pYAP300 plasmid DNA were mixed, and an equal volume of DNA Ligation Kit Ver. 2.1 (Takara Bio Inc.) Solution I was added. The mixture was incubated at 16°C for 30 minutes and then placed on ice. In this way, the genes from the cps1A gene to the cps1J gene were inserted downstream of the transcription promoter in the pYAP300 vector. The resulting vector was then added to E. coli JM109 competent cells for transformation, and the cells were plated on LB agar medium containing 500 μg / mL erythromycin and cultured at 37°C for 2 days. Plasmids were extracted from the resulting colonies, and the desired recombinant plasmid was confirmed to have been obtained based on its size and the size of the fragments produced by restriction enzyme cleavage.

[0128] Preparation Example 2 (Construction of a strain into which the cps1 gene cluster has been introduced) Using the vector for introducing the cps1 gene cluster obtained in Preparation Example 1, the cps1 gene cluster was introduced into Lacticaseibacillus casei ATCC334.

[0129] Specifically, Lacticaseibacillus casei ATCC334 was grown in MRS liquid medium, and the logarithmic growth phase culture was centrifuged at 5,000 × g for 5 minutes at 4°C to harvest the cells. The cells were washed once each with ice-cold 20 mM HEPES (pH 7.0) and 10% glycerol, and then suspended in 2 μL of 10% glycerol (Klett value of the initial culture). 50 μL of the bacterial suspension was mixed with 3 μL of the recombinant plasmid DNA solution, placed in a 2 mm-wide electroporation cuvette, and electroporated at 1.5 kV, 200 Ω, and a capacitance of 25 μF using a Gene Pulser II (Bio-Rad).

[0130] After electroporation, 1 mL of MRS liquid medium was added and the mixture was cultured at 37°C for 2 hours, after which the mixture was spread on MRS agar medium supplemented with erythromycin at a concentration of 20 μg / mL and incubated at 37°C for 2 to 3 days to obtain erythromycin-resistant colonies. Some of the resulting colonies were sampled, and insertion of the target plasmid into the desired position was confirmed by colony PCR using a primer having a sequence selected from a region near the attB site on the genome of Lacticaceae Bacillus casei ATCC334 and a primer having a sequence selected from a region relatively close to the end of the DNA prepared in Preparation Example 1.

[0131] The strain derived from Lacticaceae Bacillus casei ATCC334 obtained as described above was a cps1 gene cluster-introduced strain containing 10 genes obtained from the YIT 9029 strain, namely the cps1A gene (SEQ ID NO: 1), cps1B gene (SEQ ID NO: 2), cps1C gene (SEQ ID NO: 3), cps1D gene (SEQ ID NO: 4), cps1E gene (SEQ ID NO: 5), cps1F gene (SEQ ID NO: 6), cps1G gene (SEQ ID NO: 7), cps1H gene (SEQ ID NO: 8), cps1I gene (SEQ ID NO: 9), and cps1J gene (SEQ ID NO: 10), at the attB site on its genome.

Claims

1. A composition for imparting resistance to environmental stress to bacteria, comprising the cps1 gene cluster derived from Lacticaseibacillus paracasei.

2. The composition described in claim 1, wherein the cps1 gene group comprises the cps1A gene, the cps1B gene, the cps1C gene, the cps1D gene, the cps1E gene, the cps1F gene, the cps1G gene, the cps1H gene, the cps1I gene, and the cps1J gene.

3. The cps1A gene has the polynucleotide sequence shown in SEQ ID NO:1 or a polynucleotide sequence having 90% or more homology thereto, the cps1B gene has the polynucleotide sequence shown in SEQ ID NO:2 or a polynucleotide sequence having 90% or more homology thereto, the cps1C gene has the polynucleotide sequence shown in SEQ ID NO:3 or a polynucleotide sequence having 90% or more homology thereto, the cps1D gene has the polynucleotide sequence shown in SEQ ID NO:4 or a polynucleotide sequence having 90% or more homology thereto, the cps1E gene has the polynucleotide sequence shown in SEQ ID NO:5 or a polynucleotide sequence having 90% or more homology thereto, the cps1F gene has the polynucleotide sequence shown in SEQ ID NO:6 or a polynucleotide sequence having 90% or more homology thereto, and the cps1G gene has the polynucleotide sequence shown in SEQ ID NO:7 or a polynucleotide sequence having 90% or more homology thereto, The composition described in claim 2, wherein the cps1H gene has the polynucleotide sequence shown in SEQ ID NO: 8 or a polynucleotide sequence having 90% or more homology thereto, the cps1I gene has the polynucleotide sequence shown in SEQ ID NO: 9 or a polynucleotide sequence having 90% or more homology thereto, and the cps1J gene has the polynucleotide sequence shown in SEQ ID NO: 10 or a polynucleotide sequence having 90% or more homology thereto.

4. The cps1A gene encodes the amino acid sequence shown in SEQ ID NO: 11 or an amino acid sequence having 90% or more homology thereto, the cps1B gene encodes the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having 90% or more homology thereto, the cps1C gene encodes the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having 90% or more homology thereto, the cps1D gene encodes the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having 90% or more homology thereto, the cps1E gene encodes the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having 90% or more homology thereto, the cps1F gene encodes the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having 90% or more homology thereto, and the cps1G gene encodes the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having 90% or more homology thereto, The composition described in claim 2, wherein the cps1H gene encodes the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having 90% or more homology thereto, the cps1I gene encodes the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having 90% or more homology thereto, and the cps1J gene encodes the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having 90% or more homology thereto.

5. A composition according to any one of claims 1 to 4, wherein the cps1 gene cluster is used in a form incorporated into a vector.

6. The composition according to any one of claims 1 to 4, wherein the environmental stress to which resistance is to be imparted is environmental stress caused by at least one selected from the group consisting of acids, bile components, alcohols, metal ions, hydrophobic substances, and surfactants.

7. A method for imparting environmental stress resistance to bacteria, comprising the step of introducing the cps1 gene cluster into a target bacterium using the composition according to any one of claims 1 to 4.

8. The method for imparting environmental stress resistance according to claim 7, which imparts resistance to at least one environmental stress selected from the group consisting of acids, bile components, alcohols, metal ions, hydrophobic substances, and surfactants.

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