Lactic acid bacteria, lactic acid bacteria composition, production method for lactic acid bacteria, method for improving acid resistance of lactic acid bacteria, screening method for lactic acid bacteria, and production method for fermented milk

Abstract

A method of improving the acid resistance of lactic acid bacteria, said method including a step for artificially suppressing the function of kup genes in lactic acid bacteria.

Description

Lactic acid bacteria, lactic acid bacteria composition, method for producing lactic acid bacteria, method for improving acid tolerance of lactic acid bacteria, method for screening lactic acid bacteria, and method for producing fermented milk

[0001] The present invention relates to lactic acid bacteria, a lactic acid bacteria composition, a method for producing lactic acid bacteria, a method for improving the acid tolerance of lactic acid bacteria, a method for screening lactic acid bacteria, and a method for producing fermented milk, and more specifically to provide lactic acid bacteria and a lactic acid bacteria composition containing the same, a method for producing lactic acid bacteria, a method for improving the acid tolerance of lactic acid bacteria, a method for screening lactic acid bacteria, and a method for producing fermented milk using these lactic acid bacteria and / or lactic acid bacteria compositions.

[0002] Lactic acid bacteria are microorganisms that have long been used in the production of fermented foods such as yogurt and other fermented milks, as well as pickles. In recent years, however, they have also been attracting attention as probiotics that provide various beneficial effects to the host, such as intestinal regulation by regulating the bacterial flora in the gastrointestinal tract, improving immune function, and antitumor effects.

[0003] However, conventional lactic acid bacteria generally have low acid tolerance. For example, when ingested orally, the survival rate of lactic acid bacteria in the intestine is reduced by acids such as gastric acid and bile acid, resulting in the inability to fully exert the inherent probiotic effects of lactic acid bacteria in the intestine. Therefore, several studies aimed at improving the acid tolerance of lactic acid bacteria have been conducted. For example, Japanese Patent Laid-Open Publication No. 2005-160 (Patent Document 1) describes a method for improving the survival rate of microorganisms in the human and animal body by disrupting the hrcA gene of microorganisms, including lactic acid bacteria. Furthermore, for example, Japanese Patent Laid-Open Publication No. 2001-204468 (Patent Document 2) describes an acid-resistant lactic acid-producing microorganism containing a recombinant vector incorporating a lactate dehydrogenase gene and promoter derived from a microorganism belonging to the genus Bifidobacterium or Lactobacillus, with the aim of obtaining a microorganism with high lactic acid productivity even under acidic conditions.

[0004] JP 2005-160 A JP 2001-204468 A

[0005] However, there are still not enough reports on the factors involved in the acid resistance of lactic acid bacteria. Therefore, for example, to test whether lactic acid bacteria have excellent acid resistance, artificial gastric juice resistance tests (in vitro) and gastrointestinal transit tests using animals have been performed, but these methods have the problem that it is difficult to test a large number of lactic acid bacteria species at once. Therefore, further research on the factors involved in the acid resistance of lactic acid bacteria is desired.

[0006] The present invention has been made in consideration of the problems associated with the above-mentioned conventional technologies, and aims to provide lactic acid bacteria having sufficiently excellent resistance to acids such as gastric acid and a lactic acid bacteria composition containing the same, a method for producing lactic acid bacteria having improved acid resistance, a method for improving the acid resistance of lactic acid bacteria, a method for screening lactic acid bacteria having sufficiently excellent acid resistance, and a method for producing fermented milk using these lactic acid bacteria and / or lactic acid bacteria compositions.

[0007] The present inventors conducted extensive research to achieve the above-mentioned objective and succeeded in isolating lactic acid bacteria that exhibit a high survival rate in an acid tolerance test. Next, to explore factors involved in acid tolerance in lactic acid bacteria, genomic analysis was performed between lactic acid bacteria that exhibited a high survival rate in the acid tolerance test (highly acid-tolerant lactic acid bacteria) and conventional common lactic acid bacteria (i.e., lactic acid bacteria that exhibit a low survival rate in the acid tolerance test: low-acid-tolerant lactic acid bacteria). This genomic analysis identified frameshift mutations and nonsense mutations in the kup gene as mutations common to highly acid-tolerant lactic acid bacteria and accompanied by amino acid mutations. Furthermore, when the highly acid-tolerant lactic acid bacteria were passaged multiple times to induce mutations, lactic acid bacteria that lost acid tolerance were obtained, and genomic analysis was also performed between these and the highly acid-tolerant lactic acid bacteria. As a result, in the lactic acid bacteria that had lost their acid tolerance, the induced mutations eliminated the frameshift mutations and nonsense mutations in the kup gene extracted from the highly acid-tolerant lactic acid bacteria, and the nucleotide sequence of the kup gene was almost restored to that of the original low-acid-tolerant lactic acid bacteria.

[0008] Based on these findings, the present inventors have discovered that the kup gene is a factor involved in the acid resistance of lactic acid bacteria, and that lactic acid bacteria in which the function of the kup gene is suppressed have sufficiently excellent acid resistance. Therefore, the present inventors have discovered that the acid resistance of lactic acid bacteria can be improved by artificially suppressing the function of the kup gene, and that lactic acid bacteria with sufficiently excellent acid resistance can be easily screened using the suppression of the function of the kup gene as an indicator, leading to the completion of the present invention.

[0009] Therefore, the present invention provides the following aspects. [1] A method for improving the acid tolerance of lactic acid bacteria, comprising a step of artificially suppressing the function of the kup gene of lactic acid bacteria. [2] The method according to [1], wherein the lactic acid bacteria are lactic acid bacteria of the Lactobacillaceae family. [3] The method according to [1] or [2], wherein the lactic acid bacteria are lactic acid bacteria of the Lactobacillus genus. [4] A method for producing lactic acid bacteria with improved acid tolerance, comprising a step of artificially suppressing the function of the kup gene of lactic acid bacteria. [5] The production method according to [4], wherein the lactic acid bacteria are lactic acid bacteria of the Lactobacillaceae family. [6] The production method according to [4] or [5], wherein the lactic acid bacteria are lactic acid bacteria of the Lactobacillus genus. [7] A lactic acid bacterium in which the function of the kup gene is suppressed. [8] The lactic acid bacterium according to [7], which is a lactic acid bacterium of the family Lactobacillaceae. [9] The lactic acid bacterium according to [7] or [8], which is a lactic acid bacterium of the genus Lactobacillus.

[10] A lactic acid bacterium composition containing the lactic acid bacterium according to any one of [7] to [9].

[11] The lactic acid bacterium composition according to

[10] , which further contains a lactic acid bacterium of the genus Streptococcus.

[12] The lactic acid bacterium composition according to

[10] or

[11] , which is a lactic acid bacterium starter.

[13] The lactic acid bacterium composition according to

[10] or

[11] , which is fermented milk.

[14] A screening method for highly acid-tolerant lactic acid bacteria, comprising a selection step of selecting lactic acid bacteria using suppression of the function of the kup gene as an indicator.

[15] A method for producing fermented milk, comprising a fermentation step of adding any one selected from the group consisting of the lactic acid bacteria obtained by the production method of any one of [4] to [6], the lactic acid bacteria described in any one of [7] to [9], the lactic acid bacteria composition of any one of

[10] to

[13] , and the lactic acid bacteria selected by the screening method of

[14] to a milk preparation containing raw material milk, and fermenting the mixture to obtain fermented milk.

[16] A method for producing fermented milk according to

[15] , comprising a step of further adding Streptococcus lactic acid bacteria to the milk preparation.

[0010] According to the present invention, it is possible to provide lactic acid bacteria having sufficiently excellent resistance to acids such as gastric acid and lactic acid bacteria compositions containing the same, a method for producing lactic acid bacteria having improved acid resistance, a method for improving the acid resistance of lactic acid bacteria, a method for screening lactic acid bacteria having sufficiently excellent acid resistance, and a method for producing fermented milk using these lactic acid bacteria and / or lactic acid bacteria compositions.

[0011] The present invention will be described in detail below based on preferred embodiments thereof.

[0012] <Lactic acid bacteria> In the present invention, "lactic acid bacteria" is a general term for microorganisms that can utilize glucose to produce lactic acid in a sugar yield of 50% or more. Physiologically, they are Gram-positive cocci or bacilli that are non-motile and basically do not have the ability to form spores (with the exception of Bacillus coagulans, which has the ability to form spores), and are catalase-negative.

[0013] Examples of lactic acid bacteria according to the present invention include lactic acid bacteria of the family Streptococcusaceae, lactic acid bacteria of the family Lactobacillaceae, and lactic acid bacteria of the family Leuconostocaceae, and more specifically, lactic acid bacteria of the genus Lactobacillus, lactic acid bacteria of the genus Lacticaseibacillus, lactic acid bacteria of the genus Lactiplantibacillus, lactic acid bacteria of the genus Liquorilactobacillus, and lactic acid bacteria of the genus Limosilactobacillus. Lactobacillus lactic acid bacteria, Levilactobacillus lactic acid bacteria, Lentilactobacillus lactic acid bacteria, Weissella lactic acid bacteria, etc.; Pediococcus lactic acid bacteria, Leuconostoc lactic acid bacteria, etc. Examples of suitable lactic acid bacteria include lactic acid bacteria such as Leuconostoc lactic acid bacteria, Lactococcus lactic acid bacteria, Streptococcus lactic acid bacteria, and Enterococcus lactic acid bacteria; and Bifidobacterium lactic acid bacteria. Among these, Lactobacillaceae lactic acid bacteria are preferred, and Lactobacillus lactic acid bacteria are more preferred.

[0014] Examples of Lactobacillus lactic acid bacteria include Lactobacillus delbrueckii, Lactobacillus nasaridis, Lactobacillus equicrusolis, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus paragasseri, Lactobacillus amylovoros, and Lactobacillus crispatus. In the present invention, each species also includes its subspecies.

[0015] Among the Lactobacillus lactic acid bacteria, Lactobacillus delbrueckii is particularly preferred as the lactic acid bacterium of the present invention. Examples of Lactobacillus delbrueckii include Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. lactis, Lactobacillus delbrueckii subsp. indicus, Lactobacillus delbrueckii subsp. sunki, and Lactobacillus delbrueckii subsp. jacobsenii.

[0016] According to the present invention, not only in lactic acid bacteria but also in bifidobacteria (Bifidobacterium), the acid tolerance of bifidobacteria can be improved by artificially suppressing the function of the kup gene, and it is possible to easily screen for bifidobacteria with sufficiently excellent acid tolerance using the suppression of the function of the kup gene as an indicator. In the present invention, "bifidobacterium" is a general term for bacteria belonging to the genus Bifidobacterium in the order Bifidobacteriale of the class Actinomycetes, and is a gram-positive, obligately anaerobic rod-shaped bacterium capable of assimilating lactose and oligosaccharides to produce acetic acid and lactic acid.

[0017] (kup gene) The lactic acid bacterium of the present invention is a lactic acid bacterium in which the function of the kup gene is suppressed. The "kup gene" is a gene that is widely conserved among microorganisms, and is known as a gene that encodes a protein that functions as a potassium transporter (Kup; potassium uptake transporter) in Escherichia coli and the like. However, in lactic acid bacteria (especially Lactobacillus lactic acid bacteria), the function of the protein (or amino acid sequence) encoded by the kup gene has not yet been fully elucidated.

[0018] An example of the kup gene according to the present invention is a gene encoding the amino acid sequence set forth in SEQ ID NO: 1, and a typical nucleotide sequence thereof is set forth in SEQ ID NO: 2. The amino acid sequence set forth in SEQ ID NO: 1 and the nucleotide sequence set forth in SEQ ID NO: 2 are those of Lactobacillus delbrueckii subsp. bulgaricus P2103001 (a strain kept at the Meiji Innovation Center of Meiji Co., Ltd. (1-29-1 Shichikuni, Hachioji-shi, Tokyo, Japan, 192-0919)).

[0019] Furthermore, among lactic acid bacteria (particularly Lactobacillaceae lactic acid bacteria, preferably Lactobacillus lactic acid bacteria), the amino acid sequence encoded by the kup gene is highly conserved, and examples of the kup gene according to the present invention include genes encoding the amino acid sequences shown in Table 1 below. It should be understood that the nucleotide sequences of genes encoding these amino acids may vary at the strain level due to polymorphisms, etc. Table 1 shows the accession number (No.) of the amino acid sequence encoded by a typical kup gene of lactic acid bacteria, the amino acid identity (%) with the amino acid sequence set forth in SEQ ID NO: 1, and the name of the lactic acid bacterium from which the amino acid sequence is derived.

[0020]

[0021] (Inhibition of kup gene function) In the present invention, "inhibition of the function of the kup gene (inhibition of kup gene function)" means that a mutation accompanied by a function-inhibiting amino acid mutation has been made in the kup gene, and / or the expression of the kup gene has been reduced. Although the specific function of the amino acid sequence encoded by the kup gene of lactic acid bacteria (particularly Lactobacillus lactic acid bacteria) has not yet been fully elucidated, the present inventors have found that in lactic acid bacteria, the function of the kup gene, that is, the function of encoding and expressing at least the correct amino acid sequence (for example, the amino acid sequence described in SEQ ID NO: 1 or Table 1), can be suppressed to improve acid tolerance. Therefore, it can be said that the "function of the kup gene" in the present invention includes at least the acid-sensing function. In addition, in the present invention, when "inhibition" of a function is referred to, it also includes the loss of the function.

[0022] [Mutations involving function-inhibiting amino acid mutations] In the present invention, "function-inhibiting amino acid mutations" are not particularly limited as long as they are amino acid mutations that inhibit (including deletion of) the acid-sensing function, i.e., improve the acid tolerance of lactic acid bacteria, but include substitutions, deletions, insertions and / or additions of one or more amino acids, and more specifically, for example, substitutions, deletions, insertions and / or additions of one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1 or Table 1, or amino acid sequences corresponding thereto, and substitutions, deletions, insertions and / or additions of one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1 are preferred.

[0023] In the present invention, an amino acid sequence or region that "corresponds to" an amino acid sequence or region refers to an amino acid sequence or region that is aligned with a reference amino acid sequence or region (e.g., the amino acid sequence set forth in SEQ ID NO: 1, the amino acid sequence set forth in Table 1, or the amino acid sequence set forth in SEQ ID NO: 3 below) when the amino acid sequences are aligned using amino acid sequence analysis software (GENETYX-MAC, Sequencher, etc.) or BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) (for example, parameters: default values ​​(i.e., initial settings)).

[0024] Mutations in the kup gene that result in such function-inhibiting amino acid mutations include, for example, missense mutations, nonsense mutations, frameshift mutations, and combinations thereof, caused by substitution, deletion, insertion, and / or addition of one or more bases in the nucleotide sequence of the kup gene, and also include substitution and / or deletion of multiple or all bases due to gene knockout, etc.

[0025] Function-suppressing amino acid mutations that more sufficiently suppress the acid-sensing function, i.e., that can more sufficiently improve the acid tolerance of lactic acid bacteria, are preferably substitutions, deletions, insertions, and / or additions, preferably substitutions and / or deletions, and more preferably the number of deleted amino acids is preferably 46 or more, more preferably 60 or more, and even more preferably 70 or more. Furthermore, such function-suppressing amino acid mutations more preferably include substitutions, deletions, insertions, and / or additions in at least all or part of the C-terminal domain of the amino acid sequence encoded by the kup gene, and particularly preferably include deletion of at least all or part of the C-terminal domain.

[0026] In the present invention, the C-terminal domain of the amino acid sequence encoded by the kup gene refers to the C-terminal region from Gly at amino acid position 450 onward in the amino acid sequence shown in SEQ ID NO: 1, and the region corresponding to said region. SEQ ID NO: 3 shows a typical sequence of the C-terminal domain (from Gly at amino acid position 450 onward in the amino acid sequence shown in SEQ ID NO: 1), and SEQ ID NO: 4 shows a typical nucleotide sequence encoding this.

[0027] More specifically, the deletion of all or part of the C-terminal domain is preferably a deletion of 20% or more of the amino acids in the C-terminal domain (227 amino acids in SEQ ID NO: 3), and more preferably a deletion of 26% or more, further preferably 30% or more, 50% or more, 70% or more, 80% or more, or 100% of the amino acids.

[0028] Examples of the function-suppressing amino acid mutations include, but are not limited to, substitution and / or deletion of 46 or more amino acid residues (preferably 60 or more, or 70 or more) in the amino acid sequence set forth in SEQ ID NO: 1 or Table 1, or an amino acid sequence corresponding thereto (preferably the amino acid sequence set forth in SEQ ID NO: 1); deletion of 20% or more amino acids (preferably 26% or more, 30% or more, 50% or more, 70% or more, 80% or more, or 100%) in the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence corresponding thereto (preferably the amino acid sequence set forth in SEQ ID NO: 3);

[0029] In the present invention, methods for detecting "mutations accompanied by function-suppressing amino acid mutations" in the kup gene of lactic acid bacteria can be appropriately applied using conventionally known methods, and are not particularly limited, including, for example, the following methods. In the present invention, "detection of mutations" of a gene essentially means detecting mutations in genomic DNA, but because mutations in the genomic DNA are reflected in base changes in transcription products or amino acid changes in translation products, it also includes detecting such changes in these transcription products or translation products (i.e., indirect detection at the transcription or translation level).

[0030] In one embodiment of the detection method, the nucleotide sequence of the kup gene region is directly determined and compared with a control to detect mutations. In the present invention, the "kup gene region" refers to a certain region on genomic DNA that contains at least the kup gene, and may include, in addition to the translated region, untranslated regions such as expression control regions of the kup gene (e.g., promoter region, enhancer region) and the 5'-terminal untranslated region of the kup gene.

[0031] In this embodiment, a DNA sample is first prepared from the target lactic acid bacteria. Examples of DNA samples include genomic DNA samples and cDNA samples prepared by reverse transcription from RNA. The methods for extracting genomic DNA or RNA from the lactic acid bacteria and preparing cDNA are not particularly limited, and known methods or methods based thereon can be appropriately adopted. Next, DNA containing the kup gene region is isolated, and the nucleotide sequence of the isolated DNA is determined. The DNA can be isolated, for example, by PCR using genomic DNA or RNA as a template, using a pair of oligonucleotide primers designed to flank all or part (preferably all) of the kup gene region. Those skilled in the art can routinely design suitable oligonucleotide primers based on the nucleotide sequence of the kup gene available from public databases (e.g., Genbank) (preferably, the nucleotide sequence encoding the nucleotide sequence set forth in SEQ ID NO: 2 or the amino acid sequence set forth in Table 1). The nucleotide sequence of the isolated DNA can be determined by methods known to those skilled in the art, such as the Maxam-Gilbert method or the Sanger method. Alternatively, a DNA library may be prepared from the prepared genomic DNA or cDNA, and the nucleotide sequence of the entire genomic DNA may be determined using a next-generation sequencer or the like.

[0032] The determined nucleotide sequence of the DNA or cDNA is compared with a control. If a mutation in the gene region of the kup gene is detected in the target lactic acid bacterium, it can be determined that the function of the kup gene is suppressed depending on the type and / or degree of the mutation. The type and / or degree of the mutation in the gene region of the kup gene detected here, which can more sufficiently improve the acid tolerance of the lactic acid bacterium, is preferably the type and / or degree of the mutation involving the amino acid mutation listed as a preferred embodiment of the function-suppressing amino acid mutation. Examples of controls in this case include known nucleotide sequences of the kup gene, more specifically, nucleotide sequences encoding the nucleotide sequence set forth in SEQ ID NO: 2 or the amino acid sequence set forth in Table 1. Furthermore, in the case of the production method or acid tolerance improving method of the present invention described below, the control can be the nucleotide sequence of the kup gene of the lactic acid bacterium before the function of the kup gene is artificially suppressed.

[0033] Other embodiments of the detection method include, for example, the following methods: a method using restriction fragment length polymorphism (RFLP) or PCR-RFLP, in which DNA containing all or a part (preferably all) of the gene region of the kup gene is amplified and the size of the DNA fragments produced by restriction enzymes in the amplified product is compared with a control; a PCR-SSCP (single-strand conformation polymorphism) method, in which the amplified product is dissociated into single-stranded DNA, separated on a non-denaturing gel, and the mobility on the gel is compared with a control; and a denaturant gradient gel electrophoresis method, in which the amplified product is separated on a gel with a gradient of DNA denaturant concentration, and the mobility on the gel is compared with a control. a MALDI-TOF / MS method in which the DNA sample is used as a template to carry out a ddNTP primer extension reaction using "an oligonucleotide primer having a base on the 3' side of one base of all or part (preferably all) of the bases in the gene region of the kup gene and a nucleotide sequence complementary to the nucleotide sequence on the 3' side thereof," and the genotype determined by mass measurement is compared with that of a control; a pyrosequencing method in which the amplification product is dissociated into single strands, only one strand is separated, and an extension reaction is carried out base by base from the vicinity of all or part (preferably all) of the bases in the gene region of the kup gene, and the pyrophosphate produced during this process is measured and compared with that of a control; Examples of such methods include the AcycloPrime method, in which a single-base extension reaction is carried out using the amplification product as a template in the presence of fluorescently labeled nucleotides with "an oligonucleotide primer having a base on the 3' side of one base of all or part (preferably all) of the bases in the gene region of the kup gene and a nucleotide sequence complementary to the nucleotide sequence on the 3' side of that base," and measuring the degree of polarization of the fluorescence and comparing it with a control; and the SNuPE method, in which the type of base used in the single-base extension reaction is determined and compared with a control.

[0034] In each detection method, if the detection results obtained are different from those of a control, for example, if the result shows that the kup gene chain length is shorter than that of the control, it can be determined that the gene region of the kup gene in the lactic acid bacterium is mutated, i.e., the function of the kup gene is inhibited. In this case, the chain length of the kup gene that can more sufficiently improve the acid tolerance of the lactic acid bacterium is preferably a chain length accompanied by an amino acid mutation listed as a preferred embodiment of the function-inhibiting amino acid mutation. Examples of controls include DNA samples of the kup gene known to be free of mutations, and more specifically, standard DNA samples artificially synthesized based on the nucleotide sequence set forth in SEQ ID NO: 2 or the nucleotide sequence encoding the amino acid sequence set forth in Table 1. Furthermore, in the case of the production method or acid tolerance improving method of the present invention described below, the control can be a DNA sample extracted from lactic acid bacteria before the function of the kup gene is artificially inhibited.

[0035] [Decreased Expression of the kup Gene] In the present invention, "decreased expression of the kup gene (decreased expression of the kup gene)" means that approximately the full length of the amino acid sequence encoded by the kup gene is not expressed. Specifically, "approximately the full length of the amino acid sequence encoded by the kup gene" preferably refers to at least 70%, at least 80%, at least 90%, at least 93%, at least 95%, or at least 100% of the length of the amino acid sequence set forth in SEQ ID NO: 1 or Table 1, or an amino acid sequence corresponding thereto (preferably the amino acid sequence set forth in SEQ ID NO: 1).

[0036] The fact that approximately the entire amino acid sequence encoded by the kup gene is not expressed can be detected, for example, by Western blotting, SDS-PAGE, or amino acid sequencing.

[0037] It is also known in the art that promoter hypermethylation is one of the factors that contribute to decreased gene expression. Therefore, as another embodiment of the detection method, for example, a method of detecting expression at the transcriptional level of the kup gene using methylation of the kup gene promoter as an indicator can be employed. Promoter methylation can be detected using known methods, such as a method of directly detecting changes in the nucleotide sequence after bisulfite treatment, which has the activity of converting methylated cytosine to uracil, by nucleotide sequencing, or a method of indirectly detecting changes using a restriction endonuclease that can recognize (cleave) the nucleotide sequence before bisulfite treatment but cannot recognize (cleave) the nucleotide sequence after bisulfite treatment. Depending on the level of methylation, it can be determined that expression at the transcriptional level is not detected, i.e., that the function of the kup gene is suppressed.

[0038] In the present invention, the method for detecting "kup gene function inhibition" in lactic acid bacteria can be any one of the above detection methods, or a combination of two or more of them. Among these, from the viewpoints of accuracy and simplicity, a method for detecting kup gene function inhibition by detecting a mutation in the kup gene accompanied by a function-inhibiting amino acid mutation in the target lactic acid bacterium is preferred, and a method for detecting the mutation by directly determining the nucleotide sequence of the kup gene and comparing it with a control is more preferred.

[0039] The lactic acid bacteria of the present invention may be one species or a combination of two or more species. More specifically, examples of the lactic acid bacteria of the present invention include Lactobacillus delbrueckii (L. delbrueckii) in which the function of the kup gene is suppressed, such as Lactobacillus delbrueckii subsp. bulgaricus OLL205405 (hereinafter sometimes referred to as "strain f") identified by accession number NITE BP-03574 and L. delbrueckii subsp. bulgaricus OLL205406 (hereinafter sometimes referred to as "strain i") identified by accession number NITE BP-03575.

[0040] Strains f and i have mutations involving function-suppressing amino acid mutations that suppress the function of the kup gene. Specifically, strain f has a G to A substitution mutation at position 1850 of the nucleotide sequence set forth in SEQ ID NO: 2, accompanied by a nonsense mutation in which the codon (TGG) corresponding to Trp at position 617 of the amino acid sequence set forth in SEQ ID NO: 1 becomes a stop codon (TAG). Strain i has a T deletion mutation at position 521 of the nucleotide sequence set forth in SEQ ID NO: 2, accompanied by a frameshift mutation and nonsense mutation in which the codon corresponding to Leu at position 190 of the amino acid sequence set forth in SEQ ID NO: 1 becomes a stop codon.

[0041] Strain f has (1) identification: L. delbrueckii subsp. bulgaricus OLL205405, (2) accession number: NITE BP-03574, (3) date of accession: December 16, 2021, and strain i has (1) identification: L. delbrueckii subsp. bulgaricus OLL205406, (2) Accession number: NITE BP-03575, (3) Deposit date: December 16, 2021, respectively, (4) Depository institution: National Institute of Technology and Evaluation Patent Microorganism Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818). The Lactobacillus delbrueckii identified by these accession numbers may be a subculture of the strain, an artificial mutant strain of the strain or its subculture, a natural mutant strain, a genetically modified strain, or a derivative strain, within the range that does not inhibit the effects of the present invention, and may have a mutation other than the mutation of the kup gene accompanied by the above-mentioned function-suppressing amino acid mutation.

[0042] (Acid Tolerance) The lactic acid bacteria of the present invention are lactic acid bacteria with sufficiently excellent acid tolerance due to the suppression of the function of the kup gene (sometimes referred to herein as "highly acid-resistant lactic acid bacteria"). In the present invention, "highly acid-resistant lactic acid bacteria" refers to lactic acid bacteria that can survive even at low pH (for example, pH 3.5 or less, preferably pH 3.0 or less).

[0043] The acid resistance of lactic acid bacteria can be confirmed and evaluated, for example, by the following acid resistance test. Specifically, the target lactic acid bacteria are first activated and cultured in MRS medium at 37°C under anaerobic conditions for 18 hours, if necessary, and then cultured in MRS medium at 37°C under anaerobic conditions for 18 hours. The bacterial cells recovered after the culture are then added to artificial gastric juice (pH 3.0, 0.2% NaCl, 0.175% pepsin) and acid-treated at 37°C for 2 hours. The survival rate ((viable cell count after acid treatment / viable cell count before acid treatment) × 100 (%)) is calculated, and it can be confirmed and evaluated that the higher the survival rate, the higher the acid resistance. The "highly acid-resistant lactic acid bacteria" of the present invention may be any bacteria that can survive at the low pH, and may have a moderate survival rate. More specifically, the highly acid-resistant lactic acid bacteria preferably have a survival rate of 20% or more, more preferably 30% or more, and even more preferably 50% or more in the acid treatment test.

[0044] <Method for producing lactic acid bacteria, method for improving acid tolerance of lactic acid bacteria> Lactic acid bacteria in which the function of the kup gene is suppressed have excellent acid tolerance, and therefore, by artificially suppressing the function of the kup gene in lactic acid bacteria, it is possible to improve the acid tolerance of the lactic acid bacteria. Therefore, the present invention also provides a method for producing lactic acid bacteria with improved acid tolerance, which includes a step of artificially suppressing the function of the kup gene in lactic acid bacteria, and a method for improving the acid tolerance of lactic acid bacteria, which includes a step of artificially suppressing the function of the kup gene in lactic acid bacteria.

[0045] As described above, the kup gene that is the target of the production method or acid tolerance improvement method of the present invention typically includes a gene that encodes the amino acid sequence set forth in SEQ ID NO: 1 or Table 1. However, since the nucleotide sequence of the kup gene can change due to genetic mutation or hybridization in nature, the kup gene that is the target of the production method or acid tolerance improvement method of the present invention also includes, for example, a gene of a lactic acid bacterium that is orthologous to the gene that encodes the amino acid sequence set forth in SEQ ID NO: 1.

[0046] In the present invention, "ortholog" includes not only genes that have the same function between different species, but also genes that have the same function between different strains within the same species. More specifically, in the present invention, a "gene that is orthologous to the gene encoding the amino acid sequence set forth in SEQ ID NO: 1" refers to a "gene encoding an amino acid sequence that is most closely identical to the amino acid sequence set forth in SEQ ID NO: 1" in a certain lactic acid bacterium, which corresponds to the "gene encoding an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 1" herein. The sequence of such a gene can be obtained, for example, by obtaining the nucleotide sequence of the whole genomic DNA of the target lactic acid bacterium and searching using the amino acid sequence analysis software or BLAST, etc., with the amino acid sequence of SEQ ID NO: 1 as a query. The nucleotide sequence of the whole genomic DNA of the target lactic acid bacterium can be determined and obtained by any known method (e.g., whole genome sequencing, etc.) or, for example, from a database such as Gen Bank (NCBI).

[0047] The lactic acid bacteria that are the subject of the production method or acid tolerance improvement method of the present invention are preferably lactic acid bacteria in which the function of the kup gene is not suppressed or may be suppressed. Such lactic acid bacteria in which the function of the kup gene is suppressed or may be suppressed can be selected, for example, by the acid tolerance test described above or the screening method for lactic acid bacteria of the present invention described below.

[0048] In lactic acid bacteria, methods for artificially suppressing the function of the kup gene include, for example, introducing a mutation accompanied by a function-inhibiting amino acid mutation into the kup gene and / or reducing the expression of the kup gene. The mutation accompanied by a function-inhibiting amino acid mutation and the reduction in expression are as described above, including their preferred embodiments. As a method for artificially suppressing the function of the kup gene according to the present invention, a conventionally known method or a method similar thereto can be appropriately adopted depending on the purpose and degree of suppression.

[0049] For example, methods for introducing a mutation accompanied by a function-suppressing amino acid mutation into the kup gene include gene knockout methods; site-specific mutagenesis methods such as gene targeting using homologous recombination, the Kunkel method, and SOE-PCR; genome editing methods (such as methods using site-specific nucleases (meganucleases; zinc finger nucleases; TALENs; PPRs; CRISPR-Cas such as CRISPR-Cas9); etc.); methods of inducing mutations by repeated subculture; methods of inducing mutations by irradiation with UV, X-rays, gamma rays, etc.; methods of inducing mutations by treatment with chemical agents (e.g., nitrosoguanidine, diethylsulfate, nitrous acid); and methods of inducing mutations by exposure to gastric acid or artificial digestive fluid, and combinations of two or more of these may also be used.

[0050] Furthermore, for example, methods for reducing the expression of the kup gene include knockdown, promoter methylation, suppression of expression by expression of antisense RNA (van der Krol AR et al., Nature 333:866, 1988), and suppression of expression by RNA interference (RNAi) using double-stranded RNA having a sequence corresponding to the kup gene, and two or more of these methods may be used in combination.

[0051] In the production method or method for improving acid resistance of the present invention, it is sufficient that the acid resistance is improved compared to the lactic acid bacteria before the step of artificially suppressing the function of the kup gene is carried out, and improved acid resistance can be confirmed, for example, by the survival rate calculated in the above-mentioned acid resistance test being higher than that of the lactic acid bacteria before the step is carried out.

[0052] Furthermore, the production method or acid tolerance improving method of the present invention may further include a step of confirming that the lactic acid bacteria that have been subjected to the step of artificially suppressing the function of the kup gene are indeed the highly acid-resistant lactic acid bacteria, for example, by conducting the above-mentioned acid tolerance test.

[0053] <Screening method for lactic acid bacteria> Because lactic acid bacteria in which the function of the kup gene is suppressed have excellent acid tolerance, the present invention also provides a screening method for highly acid-tolerant lactic acid bacteria, which includes a selection step of selecting lactic acid bacteria using the suppression of the function of the kup gene as an indicator. The screening method of the present invention includes a screening method for lactic acid bacteria that are actually highly acid-resistant, as well as a screening method for lactic acid bacteria that may be highly acid-tolerant, and may also be a method for screening lactic acid bacteria at a level that is expected to have moderate acid tolerance.

[0054] In the selection step, for example, at least one of the detection methods listed above for inhibiting the function of the kup gene is used to determine whether the function of the kup gene is inhibited (i.e., whether a mutation in the kup gene accompanied by an inhibiting amino acid mutation is detected and / or whether reduced expression of the kup gene is detected), and lactic acid bacteria for which inhibited function of the kup gene is detected are selected as the highly acid-tolerant lactic acid bacteria (including moderately acid-tolerant lactic acid bacteria) or lactic acid bacteria that may be the highly acid-tolerant lactic acid bacteria. The determination method, including its preferred embodiments, is as described for each of the detection methods above.

[0055] Furthermore, the screening method of the present invention may further comprise a step of confirming that the screened lactic acid bacteria are indeed the highly acid-resistant lactic acid bacteria, for example, by carrying out the acid resistance test described above.

[0056] <Lactic acid bacteria composition> The term "lactic acid bacteria composition" refers to a composition containing lactic acid bacteria, but is not particularly limited thereto. The lactic acid bacteria composition of the present invention may contain at least the lactic acid bacteria of the present invention, and may consist solely of the lactic acid bacteria of the present invention (including a combination of two or more lactic acid bacteria of the present invention). The present invention also provides a lactic acid bacteria composition containing lactic acid bacteria produced by the method for producing lactic acid bacteria of the present invention or lactic acid bacteria selected by the screening method for lactic acid bacteria. The lactic acid bacteria composition of the present invention includes, for example, the lactic acid bacteria starter and fermented milk described below.

[0057] The lactic acid bacteria composition of the present invention may further contain other components in addition to the lactic acid bacteria of the present invention. The other components are not particularly limited, but may include, for example, solvents such as water; cultures such as the supernatant of the culture medium or medium components after the cultivation of the lactic acid bacteria has been completed; concentrates, dilutions, dried products, frozen products of the cultures, etc., and may contain one or a combination of two or more of these.

[0058] (Lactic acid bacteria starter) The lactic acid bacteria starter of the present invention contains at least the lactic acid bacteria of the present invention and can be suitably used for the method for producing fermented milk of the present invention described below. Such a lactic acid bacteria starter of the present invention may consist solely of the lactic acid bacteria of the present invention, or may further contain the other components described above, other lactic acid bacteria, yeast, bifidobacteria, etc.

[0059] The other lactic acid bacteria, yeast, and bifidobacteria include lactic acid bacteria, yeast, and bifidobacteria that have been known to be contained in fermented milk. For example, the other lactic acid bacteria include lactic acid bacteria other than the lactic acid bacteria of the present invention, and may be one or a combination of two or more of these. Among these, when the lactic acid bacteria of the present invention are Lactobacillus lactic acid bacteria, it is preferable that the lactic acid bacteria starter of the present invention further contains Streptococcus lactic acid bacteria other than the lactic acid bacteria of the present invention. It is also preferable that the lactic acid bacteria starter of the present invention contains both Lactobacillus lactic acid bacteria of the present invention and Streptococcus lactic acid bacteria of the present invention.

[0060] Lactic acid bacteria of the genus Streptococcus are aerobic gram-positive cocci, such as Streptococcus thermophilus and Streptococcus salivarius. Of these, the lactic acid bacteria of the genus Streptococcus are preferably lactic acid bacteria belonging to Streptococcus thermophilus.

[0061] The lactic acid bacteria starter of the present invention may be in the form of a liquid or a solid such as a frozen state or a dry powder, and may further contain other components, such as fermentation promoters (formic acid, nucleic acids, etc.), protective agents (saccharides), and medium components (milk, whey, etc.), and may contain one or more of these in combination.

[0062] The content of the lactic acid bacteria of the present invention in the lactic acid bacteria starter of the present invention is not particularly limited, but is preferably 0.01 to 100% by mass, more preferably 0.1 to 90% by mass. ７ cfu / g or more, preferably 1 x 10 ７ ~1 x 10 １１ cfu / g. Furthermore, when other lactic acid bacteria (preferably Streptococcus lactic acid bacteria other than the lactic acid bacteria of the present invention) are further contained, the ratio of the content of the lactic acid bacteria of the present invention to the content of the other lactic acid bacteria in the lactic acid bacteria starter is preferably 1:0.1 to 1:100, more preferably 1:1 to 1:10, in terms of the number of bacteria (converted to viable bacteria count, the same applies hereinafter). Furthermore, when the lactic acid bacteria starter of the present invention is a combination of Lactobacillus lactic acid bacteria of the present invention and Streptococcus lactic acid bacteria of the present invention, the ratio of the content of Lactobacillus lactic acid bacteria to the content of Streptococcus lactic acid bacteria in the lactic acid bacteria starter is preferably 1:0.1 to 1:100, more preferably 1:1 to 1:10, in terms of the number of bacteria (converted to viable bacteria count, the same applies hereinafter).

[0063] The lactic acid bacteria starter of the present invention may be combined with, for example, a composition containing another lactic acid bacterium (a second lactic acid bacteria composition) to form a lactic acid bacteria starter kit containing the lactic acid bacteria starter of the present invention and the second lactic acid bacteria composition. Furthermore, the lactic acid bacteria starter kit may also be combined with, for example, additives for producing fermented milk (such as the fermentation-promoting substance and protective agent), a container, instructions for use of the lactic acid bacteria starter, and the like.

[0064] (Fermented milk) The fermented milk of the present invention contains at least the lactic acid bacteria of the present invention and can be obtained, for example, by the method for producing fermented milk of the present invention described below. The present invention also provides fermented milk containing lactic acid bacteria produced by the method for producing lactic acid bacteria of the present invention or lactic acid bacteria selected by the screening method for lactic acid bacteria. The lactic acid bacteria of the present invention contained in the fermented milk of the present invention have excellent acid resistance, and therefore can reach the intestine with a high survival rate even when, for example, the fermented milk is orally ingested and exposed to acids such as gastric acid.

[0065] The fermented milk of the present invention is not particularly limited, and examples thereof include fermented milk that meets the standards for "fermented milk" set forth in the Ministerial Ordinance on Dairy Products, etc. (more specifically, fermented milk having a non-fat milk solids content of 8.0% or more and a lactic acid bacteria or yeast count (preferably the lactic acid bacteria count (more preferably the lactic acid bacteria count of the present invention); the same applies below) of 10 million / mL or more). The fermented milk of the present invention also includes fermented milk that meets the standards for "dairy lactic acid bacteria beverage" set forth in the Ministerial Ordinance on Dairy Products, etc. (more specifically, fermented milk having a non-fat milk solids content of 3.0% or more and a lactic acid bacteria or yeast count of 10 million / mL or more); and fermented milk that meets the standards for "lactic acid bacteria beverage" (more specifically, fermented milk having a non-fat milk solids content of less than 3.0% and a lactic acid bacteria or yeast count of 1 million / mL or more). The non-fat milk solids refer to the remaining components (mainly proteins, lactose, minerals, etc.) after subtracting the fat content from the total milk solids, and the number of lactic acid bacteria and yeasts refers to the number of viable bacteria measured by the test method specified in the Ministerial Ordinance on Milk, etc., which is a method using a BCP-added plate count agar medium. However, if the fermented milk is sterilized, the number is converted to the number of viable bacteria measured by the test method before sterilization.

[0066] The fermented milk of the present invention may be a fermented product obtained after the fermentation step of the method for producing fermented milk described below, or the fermented product that has been sterilized (pulverized, heated, etc.), or may be a concentrated, diluted, dried, frozen, or similar product. In the present invention, when the fermented milk has been sterilized, the number of lactic acid bacteria in the fermented milk is calculated as the viable cell count. The lactic acid bacteria contained in the fermented milk of the present invention include not only live cells but also dead cells, and also include crushed and heat-treated lactic acid bacteria, as well as concentrates, diluted, dried, and frozen products thereof. However, it is preferable that the fermented milk of the present invention contains at least live lactic acid bacteria, and it is more preferable that the lactic acid bacteria of the present invention are contained as viable cells.

[0067] The fermented milk of the present invention contains the lactic acid bacteria of the present invention and components derived from the raw milk (preferably a milk preparation) described below. Furthermore, the fermented milk may further contain other components listed in the lactic acid bacteria starter above, other lactic acid bacteria, yeast, bifidobacteria, etc., within limits that do not impair the effects of the present invention. Furthermore, the fermented milk may contain various components that can be added to foods and beverages. Further components that can be contained in fermented milk are not particularly limited, and examples thereof include sugars, sugar alcohols, minerals, vitamins, proteins, peptides, amino acids, organic acids, pH adjusters, starch and modified starch, dietary fiber, fruits and vegetables and processed products thereof, animal and plant herbal extracts, naturally occurring polymers (collagen, hyaluronic acid, chondroitin, etc.), oils and fats, thickeners, emulsifiers, solvents, surfactants, gelling agents, stabilizers, buffers, suspending agents, thickeners, excipients, disintegrants, binders, fluidizing agents, preservatives, colorants, fragrances, flavorings, sweeteners, etc., and these may be used alone or in combination of two or more.

[0068] Preferred examples of such fermented milk include yogurt, cheese, fermented cream, and fermented butter, with yogurt being particularly preferred. Specific examples of the yogurt include set-type yogurt (solid fermented milk) such as plain yogurt, soft-type yogurt (paste-like fermented milk), and drink-type yogurt (liquid fermented milk), and frozen yogurt may be made using these as ingredients. The fermented milk of the present invention can also be used as an ingredient for fermented foods such as cheese, fermented cream, fermented butter, and kefir. Furthermore, the fermented milk of the present invention can also be used as a lactic acid bacteria starter in the following method for producing fermented milk.

[0069] <Method for producing fermented milk> The method for producing fermented milk of the present invention comprises a fermentation step of adding at least one of the lactic acid bacteria of the present invention, the lactic acid bacteria produced by the method for producing lactic acid bacteria of the present invention, the lactic acid bacteria selected by the method for screening lactic acid bacteria of the present invention, and the lactic acid bacteria composition of the present invention to a milk preparation containing raw material milk, and fermenting the mixture to obtain fermented milk. When using lactic acid bacteria selected by the method for producing lactic acid bacteria or the method for screening lactic acid bacteria of the present invention, the method for producing fermented milk of the present invention may further comprise a step of artificially suppressing the function of the kup gene and the selection step, although these steps may be performed only once at the beginning.

[0070] (Milk Preparation) The milk preparation according to the present invention contains raw milk. The raw milk preferably contains lactose, and examples thereof include raw milk (e.g., milk from cows, buffaloes, sheep, goats, etc.), pasteurized milk, whole milk, skim milk, whey, and processed products thereof (e.g., whole milk powder, whole milk concentrated milk, skim milk powder, skim milk concentrated milk, condensed milk, whey powder, buttermilk, butter, cream, cheese, whey protein concentrate (WPC), whey protein isolate (WPI), α-lactalbumin (α-La), and β-lactoglobulin (β-Lg)). These may be used alone or in combination of two or more.

[0071] The milk preparation of the present invention may consist solely of the raw milk, or may be an aqueous solution, diluted solution, or concentrated solution of the raw milk. It may also contain other ingredients in addition to the raw milk, as needed. Such other ingredients include water; foods, food ingredients, or food additives such as soy milk, sugar and other sugars, sweeteners, flavorings, fruit juice, fruit pulp, vitamins, minerals, oils and fats, ceramides, collagen, milk phospholipids, and polyphenols; stabilizers, thickeners, and gelling agents such as pectin, soy polysaccharides, CMC (carboxymethylcellulose), agar, gelatin, carrageenan, and gums; and the like. These may be used alone or in combinations of two or more. The milk preparation can be prepared by mixing the ingredients while heating and / or homogenizing, as needed. Heat-sterilized or pasteurized milk preparations can also be used.

[0072] (Fermentation) The fermentation step of adding the lactic acid bacteria of the present invention or the lactic acid bacteria composition of the present invention to the prepared milk and fermenting it can be carried out by any conventionally known method, and is not particularly limited. The amount of the lactic acid bacteria of the present invention or the lactic acid bacteria composition of the present invention added can be appropriately determined according to the amount of a lactic acid bacteria starter used in conventionally known methods for producing fermented milk. For example, the number of viable cells of the lactic acid bacteria of the present invention relative to the mass of the prepared milk can be set to 1 x 10 ７ ~5 x 10 ９ cfu / g, preferably 1 x 10 ８ ~2 x 10 ９ More preferably, it is cfu / g.

[0073] In addition to the lactic acid bacteria or lactic acid bacteria composition of the present invention, the above-mentioned other lactic acid bacteria, yeast, bifidobacteria, etc. may be added to the prepared milk. Among these, when the lactic acid bacteria of the present invention are Lactobacillus lactic acid bacteria, in the method for producing fermented milk of the present invention, it is preferable to further add Streptococcus lactic acid bacteria as the other lactic acid bacteria, and it is more preferable to further add Streptococcus thermophilus lactic acid bacteria. Furthermore, when other lactic acid bacteria (preferably Streptococcus lactic acid bacteria) are further added, the ratio of the amount of the lactic acid bacteria of the present invention added to the amount of the other lactic acid bacteria added is preferably 1:0.1 to 1:100, more preferably 1:1 to 1:10, in terms of the number of viable bacteria (hereinafter the same). Furthermore, when the lactic acid bacteria of the present invention are a combination of Lactobacillus lactic acid bacteria, which are the lactic acid bacteria of the present invention, and Streptococcus lactic acid bacteria, which are the lactic acid bacteria of the present invention, the ratio of the amount of Lactobacillus lactic acid bacteria added to the amount of Streptococcus lactic acid bacteria added is preferably 1:0.1 to 1:100 in terms of the number of bacteria (converted to the number of viable bacteria, the same applies below), and more preferably 1:1 to 1:10.

[0074] The fermentation conditions can be appropriately selected depending on the growth conditions of the lactic acid bacteria to be added, the amount of the milk preparation, etc., and are not particularly limited. For example, it is preferable to leave the milk preparation to stand or stir (preferably leave it to stand) under aerobic or anaerobic conditions at a temperature of 35 to 45°C, more preferably 38 to 43°C, until the pH of the milk preparation to which the lactic acid bacteria of the present invention or the lactic acid bacteria composition of the present invention has been added reaches 4.7 or less, more preferably 4.6 or less, and even more preferably 4.0 to 4.5, usually for 2 to 24 hours, more preferably 3 to 8 hours, and even more preferably 4 to 6 hours.

[0075] The fermented milk of the present invention can be obtained by the above-mentioned fermentation. The fermented product after the fermentation step can be used as is, or by concentrating, diluting, drying, freezing, or the like as needed to produce the fermented milk of the present invention. The fermented milk of the present invention can also be produced by crushing or heat-treating the lactic acid bacteria in the fermented product, or by concentrating, diluting, drying, freezing, or the like as needed. Therefore, the method for producing fermented milk of the present invention may further include these steps (concentrating step, diluting step, drying step, freezing step, crushing step, heat-treating step, etc.).

[0076] The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. In each of the following test examples, "%" indicates weight / volume (w / v: g / mL) percentage unless otherwise specified.

[0077] (Test Example 1) (1) The following three strains of lactic acid bacteria were used: Strain P2103001 (Comparative Example 1): Lactobacillus delbrueckii subsp. bulgaricus, a strain stored at Meiji Innovation Center of Meiji Co., Ltd. (1-29-1 Shichikuni, Hachioji City, Tokyo, Japan, Postal Code 192-0919); Strain f (Example 1): Lactobacillus delbrueckii subsp. bulgaricus identified by Accession No. NITE BP-03574; Strain i (Example 2): Lactobacillus delbrueckii subsp. bulgaricus identified by Accession No. NITE BP-03575.

[0078] (2) Acid Tolerance Test Each lactic acid bacterial strain (P2103001, f, i) was stimulated and cultured in MRS medium at 37°C under anaerobic conditions for 18 hours. 50 μL of the bacterial solution after the stimulated culture was inoculated into a small test tube containing 5 mL of MRS medium and further cultured at 37°C under anaerobic conditions for 18 hours. After further culturing, the culture was centrifuged at 3000 rpm and 4°C for 10 minutes to recover the precipitate (cell pellet). The recovered precipitate was suspended in 0.85% saline and centrifuged again under the above conditions. The supernatant was removed (washing) twice. 5 mL of 0.85% saline was added to the precipitate after the supernatant was removed to obtain a cell suspension. In addition, the pH of an aqueous solution containing 0.2% NaCl and 0.175% pepsin (1:10,000, Fujifilm Wako Pure Chemical Industries, Ltd.) was adjusted with hydrochloric acid (1N) and filtered to prepare an artificial gastric juice with a pH of 3.0.

[0079] One mL of the bacterial cell suspension was added to 9 mL of the artificial gastric fluid, and the mixture was treated with acid at 37°C for 2 hours. One mL of the solution after acid treatment was sampled and neutralized with 9 mL of 67 mM phosphate buffer (pH 6.5) to obtain an acid-treated bacterial solution. The bacterial cell suspension and the acid-treated bacterial solution were each serially diluted with PBS, and 100 μL of each was smeared on an agar medium and cultured at 37°C under anaerobic conditions for 48 hours. The viable cell count in each solution was determined from the number of colonies observed after culture and the dilution ratio, and the viable cell count was calculated using the following formula: Survival rate (%) = (viable cell count in acid-treated bacterial solution × 100 / viable cell count in bacterial cell suspension) × 100. As a result, the survival rate of strain P2103001 was 2.58%, which was similar to that of conventional lactic acid bacteria, while the survival rate of strain f was 65.8% and the survival rate of strain i was 95.4%, indicating that strains f and i are highly resistant to artificial gastric juice (acid).

[0080] (3) Draft Genome Sequencing [Genomic DNA Extraction] Each lactic acid bacteria strain (P2103001 strain, strain f, strain i) was cultured in MRS medium at 37 ° C under anaerobic conditions for 18 hours, and then 1 mL of the culture solution was taken and centrifuged at 13,000 g for 2 minutes at room temperature to recover the precipitate (cell pellet). The recovered precipitate was suspended in 10 mM Tris-HCl (pH 8.0) and centrifuged again under the above conditions. The supernatant was removed (washed) twice, and genomic DNA was extracted using a Wizard Genomic DNA Purification Kit (Promega). The DNA concentration was measured using Qubit (Thermo Fisher Scientific) and diluted with ultrapure water to 0.2 ng / μL to obtain a genomic DNA sample.

[0081] [Draft genome acquisition using Miseq] First, a DNA library was prepared from the genomic DNA sample obtained above using a Nextera XT DNA Library Prep kit (manufactured by Illumina) and purified using an AMPure XP (manufactured by Beckman Coulter Life Sciences). The average library size was measured using a High Sensitivity DNA kit (manufactured by Agilent), and the concentration was measured using a Quan-iT dsDNA High Sensitivity Assay kit (manufactured by Thermo Fisher Scientific). Next, the DNA was diluted with ultrapure water to a concentration of 2 nM, and all samples were mixed in equal amounts. The resulting mixture was denatured by adding 0.2 N sodium hydroxide solution. Next, 10 μL of the denatured mixture (denatured DNA) was mixed with 990 μL of HT1 buffer (manufactured by Illumina), and then an equal amount of 0.2 pM PhiX (manufactured by Illumina) was added. Sequence analysis was performed using a next-generation sequencer, Miseq sequencer (manufactured by Illumina), using Miseq Reagent Kit v3 (manufactured by Illumina, 600 cycles).

[0082] [Data Analysis] The sequence data obtained above was transferred to CLC Genomics Workbench (QIAGEN), contigs were created, and annotations were added. The contig of strain P2103001, which had a low survival rate in the acid tolerance test in (2) above, was used as the reference sequence, and the contigs of strains f and i, which had high survival rates, were attached to detect genetic mutations, and only mutations associated with amino acid mutations from strain P2103001 were extracted. Table 2 below shows the mutations from strain P2103001 confirmed in strains f and i. Table 2 also shows the survival rates of each of the above strains.

[0083]

[0084] As shown in Table 2, strains f and i, which are highly acid-resistant, had seven and nine mutations (non-synonymous substitutions) associated with amino acid mutations from strain P2103001, respectively. Of these, the only mutation common to the two strains was a mutation in the kup gene. The nucleotide sequence of the kup gene of strain P2103001 is shown in SEQ ID NO: 2, and the amino acid sequence encoded by this nucleotide sequence is shown in SEQ ID NO: 1. In strain f, G at position 1850 in the nucleotide sequence of strain P2103001 was replaced with A, and the codon corresponding to Trp at position 617 in the amino acid sequence of strain P2103001 was replaced with a stop codon (nonsense mutation, Table 4 below). Furthermore, in strain i, T at position 521 in the nucleotide sequence of strain P2103001 was deleted, and a frameshift mutation substituted the amino acids following Phe at position 174 in the amino acid sequence of strain P2103001. Furthermore, the codon corresponding to Leu at position 190 (position 16 from Phe174) in the amino acid sequence of strain P2103001 was replaced with a stop codon (Table 6 below). In Table 2, "transposase-related" indicates a gene encoding a protein related to transposase, "unannotated" indicates a gene for which information cannot be identified, and "lysA" indicates a gene encoding phage-derived lyzozyme. It can be said that all of these genes do not affect acid resistance. Therefore, the kup gene was considered to be a factor involved in acid resistance.

[0085] (Test Example 2) Analysis of revertant strains [Strain f] Strain f was cultured in a 10% skim milk powder medium containing 10% by weight skim milk powder (10 parts by weight skim milk powder and 90 parts by weight distilled water) and 0.1% Yeast Extract (Gibco) at 37 ° C. under anaerobic conditions for 18 hours, and 50 μL of the cultured bacterial solution was inoculated into 5 mL of 10% skim milk powder medium and further cultured under the above conditions (passage) repeatedly to induce mutation. After the 10th passage, an acid resistance test was performed in the same manner as in Test Example 1 (2), and a strain (f-1) with a significantly reduced survival rate of 0.74% was obtained.

[0086] Furthermore, strain f was cultured in MRS medium at 37°C under anaerobic conditions for 18 hours, and 50 μL of the cultured bacterial solution was inoculated into 5 mL of MRS medium and further cultured under the above conditions (passage) for 10 times to induce mutation. The strain from the 10th passage, designated strain f-2, was subjected to an acid resistance test in the same manner as in Test Example 1 (2). The survival rate was 37.7%, indicating that acid resistance was maintained.

[0087] For the obtained strains f-1 and f-2, draft genome sequencing was performed to detect gene mutation sites in the same manner as in Test Example 1 (3), except that the contig of strain f was used as the reference sequence, and only mutation sites associated with amino acid mutations from strain f were extracted. Table 3 below shows the mutations from strain f confirmed in strains f-1 and f-2. Table 3 also shows the survival rate of each of the above strains. Table 4 below shows details of the mutation at amino acid 617 (nucleotides 1849 to 1851) in strain P2103001, strain f, strain f-1, and strain f-2.

[0088]

[0089]

[0090] As shown in Tables 3-4, strains f-1 and f-2 had nine and six mutations (non-synonymous substitutions) associated with amino acid mutations from strain f, respectively. Of these, the only mutations other than the above-mentioned "transposase-related," "unannotated," and "lysA" were mutations in the kup gene in strain f-1. In strain f-1, which had reduced acid resistance, T at position 1849 in the nucleotide sequence of strain f was replaced with C in the kup gene, and the stop codon of strain f was replaced with Gln. Therefore, compared to the amino acid sequence of strain P2103001, strain f-1 only had a mutation (one amino acid substitution) in which Trp at position 617 was replaced with Gln. On the other hand, in strain f-2, which maintained acid resistance, no mutations associated with amino acid mutations from strain f were confirmed in the kup gene. In other words, it was shown that acid sensitivity was restored and acid tolerance was reduced in strain f-1 due to the elimination of the nonsense mutation in the kup gene in strain f. Therefore, it was confirmed that the kup gene is a factor involved in acid tolerance, and that acid tolerance is improved by suppressing its function.

[0091] [Strain i] Strain i was cultured in MRS medium at 37°C under anaerobic conditions for 18 hours, and 50 μL of the cultured bacterial solution was inoculated into 5 mL of MRS medium and further cultured under the same conditions (passage). After the 10th passage, an acid resistance test was performed in the same manner as in (2) of Test Example 1, and a strain (i-1) was obtained with a significantly reduced survival rate of 0.074%.

[0092] For the obtained strain i-1, draft genome sequencing was performed to detect gene mutations in the same manner as in Test Example 1 (3), except that the contig of strain i was used as the reference sequence, and only mutations associated with amino acid mutations from strain i were extracted. Table 5 below shows the mutations from strain i confirmed in strain i-1. Table 5 also shows the survival rate of strain i-1. Table 6 below also shows details of the mutations at positions 172 to 190 of the amino acid sequence and positions 514 to 525 of the nucleotide sequence (base sequence) corresponding to positions 172 to 175 of the amino acid sequence in strain P2103001, strain i, and strain i-1, as well as the sequence numbers indicating positions 172 to 190 of each amino acid sequence (positions 172 to 189 for strain i).

[0093]

[0094]

[0095] As shown in Tables 5-6, strain i-1 had 18 mutations (non-synonymous substitutions) associated with amino acid mutations from strain i. Of these, the only mutations other than the above-mentioned "transposase-related," "unannotated," and "lysA" were mutations in the kup gene. In strain i-1, which had reduced acid tolerance, an A was inserted at position 516 of the nucleotide sequence of strain i in the kup gene, resulting in the substitution of Ala at position 173 with Gly, and a frameshift mutation, restoring the amino acid sequence to the same amino acid sequence as the amino acid sequence from position 174 onwards of the amino acid sequence of strain P2103001. In other words, in strain i-1, the frameshift mutation in the kup gene in strain i was eliminated, indicating that acid sensitivity was restored and acid tolerance was reduced. Therefore, this also confirmed that the kup gene is a factor involved in acid tolerance, and that suppressing its function improves acid tolerance.

[0096] As explained above, according to the present invention, it is possible to provide lactic acid bacteria having sufficiently excellent resistance to acids such as gastric acid and lactic acid bacteria compositions containing the same, a method for producing lactic acid bacteria having improved acid resistance, a method for improving the acid resistance of lactic acid bacteria, a method for screening lactic acid bacteria having sufficiently excellent acid resistance, and a method for producing fermented milk using these lactic acid bacteria and / or lactic acid bacteria compositions.

[0097] 1. (1) Identification: L. delbrueckii subsp. bulgaricus OLL205405 (2) Accession number: NITE BP-03574 (3) Date of deposit: December 16, 2021 (4) Depository: National Institute of Technology and Evaluation, Patent Microorganisms Depositary 2. (1) Identification: L. delbrueckii subsp. bulgaricus OLL205406 (2) Accession number: NITE BP-03575 (3) Date of deposit: December 16, 2021 (4) Depository: National Institute of Technology and Evaluation, Patent Microorganisms Depositary

Claims

1. A method for improving the acid tolerance of lactic acid bacteria, comprising the step of artificially suppressing the function of the kup gene of lactic acid bacteria.

2. The method according to claim 1, wherein the lactic acid bacteria are Lactobacillaceae lactic acid bacteria.

3. The method according to claim 1, wherein the lactic acid bacteria are Lactobacillus lactic acid bacteria.

4. A method for producing lactic acid bacteria with improved acid tolerance, which comprises a step of artificially suppressing the function of the kup gene of lactic acid bacteria.

5. The method according to claim 4, wherein the lactic acid bacteria are Lactobacillaceae lactic acid bacteria.

6. The method according to claim 4, wherein the lactic acid bacteria are Lactobacillus lactic acid bacteria.

7. Lactic acid bacteria in which the function of the kup gene is suppressed.

8. The lactic acid bacterium according to claim 7, which is a Lactobacillaceae lactic acid bacterium.

9. The lactic acid bacterium according to claim 7, which is a Lactobacillus lactic acid bacterium.

10. A lactic acid bacteria composition containing the lactic acid bacteria according to claim 7.

11. The lactic acid bacteria composition according to claim 10, further comprising Streptococcus lactic acid bacteria.

12. The lactic acid bacteria composition according to claim 10, which is a lactic acid bacteria starter.

13. The lactic acid bacteria composition according to claim 10, which is fermented milk.

14. A screening method for highly acid-tolerant lactic acid bacteria, comprising a selection step of selecting lactic acid bacteria using suppression of the function of the kup gene as an indicator.

15. A method for producing fermented milk, comprising a fermentation step of adding any one selected from the group consisting of lactic acid bacteria obtained by the production method of any one of claims 4 to 6, lactic acid bacteria described in any one of claims 7 to 9, a lactic acid bacteria composition described in any one of claims 10 to 13, and lactic acid bacteria selected by the screening method of claim 14 to a milk preparation containing raw material milk, and fermenting the mixture to obtain fermented milk.

16. The method for producing fermented milk according to claim 15, further comprising the step of adding Streptococcus lactic acid bacteria to the prepared milk.