Oxygen-tolerant bacterium of genus bifidobacterium, culture method and screening method for said bacterium, composition containing oxygen-tolerant bacterium of genus bifidobacterium, and method for producing said composition

Abstract

The present invention addresses the problem of providing: an oxygen-tolerant bacterium of the genus Bifidobacterium, which can be cultured even under aerobic conditions; a culture method and a screening method for the bacterium; a composition containing oxygen-tolerant bacterium of the genus Bifidobacterium; and a method for producing the composition.　The present invention which can solve the problem is a method for culturing a bacterium of the genus Bifidobacterium, wherein the bacterium has any one gene selected from the below-mentioned genes (1) to (7). The method includes culturing the bacterium under aerobic conditions. (1) A gene which comprises a nucleotide sequence represented by SEQ ID NO: 1 or 2. (2) A gene which has 90% or higher identity with the nucleotide sequence represented by SEQ ID NO: 1 or 2. (3) A gene which comprises a nucleotide sequence having a structure such that one to several nucleotides are deleted, substituted, or added in the nucleotide sequence represented by SEQ ID NO: 1 or 2. (4) A gene which comprises a nucleotide sequence of a DNA fragment that is capable of hybridizing, under stringent conditions, with a DNA fragment comprising a sequence complementary to the nucleotide sequence represented by SEQ ID NO: 1 or 2. (5) A gene which comprises a degenerate isomer of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (6) A gene which encodes a protein comprising an amino acid sequence represented by SEQ ID NO: 3 or 4. (7) A gene which encodes a protein comprising an amino acid sequence having a structure such that one to several amino acid residues are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 3 or 4.

Description

Oxygen-tolerant Bifidobacterium bacteria, methods for culturing and screening for the same, and compositions containing oxygen-tolerant Bifidobacterium bacteria and methods for producing the same

[0001] The present invention relates to oxygen-tolerant Bifidobacterium bacteria, methods for culturing and screening for the same, and compositions containing oxygen-tolerant Bifidobacterium bacteria and methods for producing the same.

[0002] Bifidobacteria, a type of intestinal bacteria, have been found to have various beneficial effects on human health and are used in foods, beverages, and pharmaceuticals. Many pharmaceuticals and foods use bifidobacteria, particularly in dairy products such as fermented milk (yogurt). However, bifidobacteria are obligate anaerobes that require anaerobic conditions for survival, making them difficult to handle in an aerobic environment.

[0003] In light of these issues, a strain of bacteria with excellent aerobic growth was isolated from the feces of healthy breastfed infants (Bifidobacterium breve SBR3212 (Bifidobacterium Research Institute Bacteria No. 11915)), and its use as a starter for fermented milk has been proposed (Patent Document 1).

[0004] Japanese Patent Application Publication No. 04-320642

[0005] Salvatore Cosentino, Wataru Iwasaki, “SonicParanoid2: fast, accurate, and comprehensive orthology inference with machine learning and language models”, bioRxiv 2023.05.14.540736; doi: https: / / doi. org / 10.1101 / 2023.05.14.540736Yu T, Cui H, Li JC, Luo Y, Jiang G, Zhao H. , “Enzyme function prediction using contrastive learning.”, Science. 2023 Mar 31;379(6639):1358-1363. doi:10.1126 / science. adf2465. Epub 2023 Mar 30. , PMID:36996195 Kozakai T, Nakajima A, Miyazawa K, Sasaki Y, Odamaki T, Katoh T, Fukuma T, Xiao JZ, Suzuki T, Katayama T, Sakanaka M. “An improved temperature-sensitive shuttle vector system for scarless gene deletion in human-gut-associated Bifidobacterium species.”, iScience. 2024 Oct 1;27(11):111080. doi:10.1016 / j. isci. 2024.111080. PMID: 39502284; PMCID: PMC11536034.

[0006] Patent Document 1 discloses oxygen-tolerant Bifidobacterium bacteria that can be cultivated under aerobic conditions. However, it was unclear what factors Bifidobacterium bacteria, which are originally known as obligate anaerobes, must possess to exhibit oxygen tolerance.

[0007] In view of these problems, an object of the present invention is to provide oxygen-tolerant Bifidobacterium bacteria, a method for culturing and screening for the same, and a composition containing oxygen-tolerant Bifidobacterium bacteria and a method for producing the same.

[0008] As a result of extensive research and efforts, the present inventors have demonstrated that Bifidobacterium bacteria having a gene consisting of the base sequence shown in SEQ ID NO: 1 or 2 have oxygen resistance, and have completed the present invention. The present invention, which solves the above-mentioned problems, is as follows.

[0009] [1] A method for culturing Bifidobacterium bacteria having any one gene selected from the following (1) to (7), under aerobic conditions: (1) a gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2; (2) a gene having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 or 2; (3) a gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2, in which one to several nucleotides have been deleted, substituted, or added; (4) a gene consisting of a DNA nucleotide sequence that hybridizes under stringent conditions with DNA consisting of the complementary sequence of the nucleotide sequence represented by SEQ ID NO: 1 or 2; (5) a gene consisting of a degenerate isomer of the nucleotide sequence represented by SEQ ID NO: 1 or 2; (6) a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4; (7) a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4, in which one to several amino acids have been deleted, substituted, or added.

[0010] [2] The culture method according to [1], wherein the Bifidobacterium bacterium is Bifidobacterium longum.

[0011] [3] The culture method according to [1], wherein the Bifidobacterium bacterium is Bifidobacterium longum subsp. infantis.

[0012] [4] The culture method according to [1], wherein the Bifidobacterium bacterium is Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068).

[0013] [5] A method for producing a composition containing Bifidobacterium bacteria, comprising the culture method according to any one of [1] to [4].

[0014] [6] The method according to [5], further comprising a step of drying a bacterial liquid containing the Bifidobacterium bacteria cultured by the culture method, wherein the composition is a bacterial powder obtained by the drying or a food composition to which the bacterial powder has been added.

[0015] [7] The production method according to [5], comprising a step of adding a starter culture containing the Bifidobacterium bacteria cultured by the culture method to a raw material composition and fermenting it.

[0016] [8] A modified Bifidobacterium bacterium having enhanced or imparted oxygen tolerance, which is obtained by introducing any one gene selected from (1) to (7) above.

[0017] [9] A method for screening for oxygen-tolerant Bifidobacterium bacteria, comprising a step of selecting Bifidobacterium bacteria using any one gene selected from (1) to (7) above as an indicator.

[0018]

[10] A composition for starter culture, comprising a Bifidobacterium bacterium having any one gene selected from (1) to (7).

[0019]

[11] A composition comprising a Bifidobacterium bacterium having any one gene selected from (1) to (7) above, and one or more lactic acid bacteria selected from Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus delbrueckii.

[0020] According to the present invention, it is possible to provide oxygen-tolerant Bifidobacterium bacteria, methods for culturing and screening for the same, as well as a composition containing the Bifidobacterium bacteria and a method for producing the same.

[0021] Preferred embodiments of the present invention will be described below. However, the present invention is not limited to the following preferred embodiments and can be freely modified within the scope of the present invention. In this specification, percentages are expressed by mass unless otherwise specified.

[0022] <Culturing Method> The culturing method of the present invention is characterized by comprising a step of culturing, under aerobic conditions, a Bifidobacterium bacterium having any one gene selected from the following (1) to (7): (1) The gene consisting of the base sequence represented by SEQ ID NO: 1 or 2 was discovered by comparative genome analysis in the test example described below.

[0023] (1) A gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (2) A gene having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 or 2. (3) A gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2, in which one to several nucleotides are deleted, substituted, or added. (4) A gene consisting of a DNA nucleotide sequence that hybridizes under stringent conditions with DNA consisting of a complementary sequence to the nucleotide sequence represented by SEQ ID NO: 1 or 2. (5) A gene consisting of a degenerate isomer of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (6) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4. (7) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4, in which one to several amino acids are deleted, substituted, or added.

[0024] The base sequences represented by SEQ ID NOs: 1 and 2 and the amino acid sequences represented by SEQ ID NOs: 3 and 4 are as shown in the table below.

[0025]

[0026] Bifidobacterium bacteria having any one gene selected from (1) to (7) are oxygen-tolerant and can therefore be cultured under aerobic conditions.

[0027] Hereinafter, for convenience of explanation, "any one gene selected from (1) to (7)" may be referred to as "the gene of the present invention." Furthermore, "a Bifidobacterium bacterium having any one gene selected from (1) to (7)" may be referred to as "the Bifidobacterium bacterium of the present invention."

[0028] The Bifidobacterium may be obtained from animals, including humans, or from the environment, or may be artificially produced by introducing the gene of the present invention using genetic engineering techniques.

[0029] The Bifidobacterium bacteria of the present invention may be established strains of bacteria with the same genetic background, or may be a bacterial population that does not have the same genetic background but shares the same gene as long as it contains the gene of the present invention.

[0030] The Bifidobacterium genus bacteria of the present invention are not particularly limited, and examples thereof include Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium adolescentis, Bifidobacterium animalis, and Bifidobacterium pseudolongum. Examples of the Bifidobacterium include Bifidobacterium pseudolongum, Bifidobacterium reuteri, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, and Bifidobacterium lactis.

[0031] In a preferred embodiment of the present invention, the Bifidobacterium is Bifidobacterium longum.

[0032] More specifically, the present Bifidobacterium bacterium is preferably Bifidobacterium longum subsp. infantis.

[0033] More specifically, the Bifidobacterium bacterium of the present invention is preferably Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068).

[0034] In one embodiment, the Bifidobacterium bacterium does not include Bifidobacterium breve SBR3212 (Bifidobacterium sp. strain no. 11915).

[0035] [Gene] (1) Gene consisting of the base sequence represented by SEQ ID NO: 1 or 2 The gene consisting of the base sequence represented by SEQ ID NO: 1 is a gene encoding SDR (NADH oxidoreductase). The gene consisting of the base sequence represented by SEQ ID NO: 2 is a gene encoding a transcriptional regulator. The above two types of genes are genes commonly possessed by oxygen-tolerant Bifidobacterium strains that can be cultivated under aerobic conditions, and are concluded to be involved in oxygen tolerance. Therefore, Bifidobacterium bacteria having a gene consisting of the base sequence represented by SEQ ID NO: 1 or 2 have oxygen tolerance that allows them to be cultivated under aerobic conditions.

[0036] The Bifidobacterium bacterium may have either a gene consisting of the base sequence shown in SEQ ID NO: 1 or a gene consisting of the base sequence shown in SEQ ID NO: 2, or may have both.

[0037] (2) A gene having 90% or more identity to the base sequence represented by SEQ ID NO: 1 or 2 The Bifidobacterium bacterium of the present invention may have either a gene having 90% or more identity to the base sequence represented by SEQ ID NO: 1 or a gene having 90% or more identity to the base sequence represented by SEQ ID NO: 2, or may have both.

[0038] In the gene (2) above, the identity is preferably 93% or more, more preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more.

[0039] The gene (2) is preferably a gene that has 90% or more identity with the base sequence represented by SEQ ID NO: 1 or 2 and has the function of enhancing or imparting oxygen tolerance when introduced into a Bifidobacterium bacterium.

[0040] (3) A gene consisting of a base sequence in which one to several bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 1 or 2 The Bifidobacterium bacterium of this invention may have either a gene consisting of a base sequence in which one to several bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 1, or a gene consisting of a base sequence in which one to several bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 2, or it may have both.

[0041] In the gene (3), the number of deletions, substitutions, or additions of bases is preferably 1 to 100, more preferably 1 to 90, even more preferably 1 to 70, even more preferably 1 to 50, even more preferably 1 to 20, even more preferably 1 to 10, and even more preferably 1 to 5.

[0042] The gene (3) is preferably a gene that has a base sequence in which one to several bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 1 or 2, and that has the function of enhancing or imparting oxygen tolerance when introduced into a bacterium of the genus Bifidobacterium.

[0043] (4) A gene consisting of a DNA base sequence that hybridizes under stringent conditions with DNA consisting of a complementary sequence of the base sequence shown in SEQ ID NO: 1 or 2 The Bifidobacterium bacterium in question may have either one of the following forms, or both: a gene consisting of a DNA base sequence that hybridizes under stringent conditions with DNA consisting of a complementary sequence of the base sequence shown in SEQ ID NO: 1; or a gene consisting of a DNA base sequence that hybridizes under stringent conditions with DNA consisting of a complementary sequence of the base sequence shown in SEQ ID NO: 2.

[0044] As used herein, "under stringent conditions" means hybridization under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by those skilled in the art, for example, based on the length of the DNA. Basic conditions are set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001. Preferably, moderately stringent conditions include hybridization conditions of 1xSSC to 6xSSC and 42°C to 55°C, more preferably 1xSSC to 3xSSC and 45°C to 50°C, and most preferably 2xSSC and 50°C. When the hybridization solution contains, for example, about 50% formamide, a temperature 5 to 15°C lower than the above temperature is used. Washing conditions include 0.5xSSC to 6xSSC and 40°C to 60°C. Generally, 0.05% to 0.2%, preferably about 0.1%, SDS may be added during hybridization and washing. Highly stringent conditions can also be easily determined by those skilled in the art, for example, based on the length of the DNA. Generally, highly stringent (highly stringent) conditions involve hybridization and / or washing at a higher temperature and / or lower salt concentration than moderately stringent conditions. For example, hybridization conditions include 0.1xSSC to 2xSSC and 55°C to 65°C, more preferably 0.1xSSC to 1xSSC and 60°C to 65°C, and most preferably 0.2xSSC and 63°C. Washing conditions include 0.2xSSC to 2xSSC, 50°C to 68°C, and more preferably 0.2xSSC, 60°C to 65°C.

[0045] The gene (4) is preferably a gene that has a base sequence of DNA that hybridizes under stringent conditions with DNA consisting of a complementary sequence of the base sequence represented by SEQ ID NO: 1 or 2, and that has the function of enhancing or imparting oxygen resistance when introduced into a Bifidobacterium bacterium.

[0046] (5) A gene consisting of a degenerate isomer of the base sequence represented by SEQ ID NO: 1 or 2 The Bifidobacterium bacterium of the present invention may have either a gene consisting of a degenerate isomer of the base sequence represented by SEQ ID NO: 1 or a gene consisting of a degenerate isomer of the base sequence represented by SEQ ID NO: 2, or may have both.

[0047] (6) Gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4. The protein consisting of the amino acid sequence represented by SEQ ID NO: 3 is SDR (NADH oxidoreductase). The protein consisting of the amino acid sequence represented by SEQ ID NO: 4 is a transcriptional regulator.

[0048] The Bifidobacterium bacterium may have either a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4, or may have both.

[0049] (7) A gene encoding a protein consisting of an amino acid sequence in which one to several amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 3 or 4 The Bifidobacterium bacterium of the present invention may have either a gene encoding a protein consisting of an amino acid sequence in which one to several amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 3, or a gene encoding a protein consisting of an amino acid sequence in which one to several amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 4, or it may have both.

[0050] In the gene of (7), the number of the amino acids deleted, substituted, or added is preferably 1 to 30, more preferably 1 to 25, even more preferably 1 to 20, even more preferably 1 to 15, even more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3.

[0051] The gene (7) is preferably a gene that encodes a protein consisting of an amino acid sequence in which one to several amino acids are deleted, substituted, or added in the amino acid sequence represented by SEQ ID NO: 3 or 4, and that has the function of enhancing or imparting oxygen tolerance when introduced into a bacterium of the genus Bifidobacterium.

[0052] [Culturing under aerobic conditions] The culturing method of the present invention includes a step of culturing the present Bifidobacterium bacteria under aerobic conditions.

[0053] "Aerobic conditions" refer to conditions under which aerobic bacteria can perform aerobic respiration. Specifically, this includes not only aerobic environments in which oxygen is present in the culture environment, but also anaerobic environments in which oxygen is absent in the culture environment but where a certain concentration of dissolved oxygen is present in the medium. In the culture method of the present invention, examples of aerobic conditions include conditions in which the oxygen concentration in the culture environment is preferably 1% or higher, more preferably 3% or higher, and even more preferably 6% or higher. The oxygen concentration in the culture environment can be, for example, 21% or lower, preferably 18% or lower, and more preferably 12% or lower. In the culture method of the present invention, adjusting the oxygen concentration in the culture environment as described above eliminates the need for conventional culture involving the aeration of anaerobic gases such as carbon dioxide. The culture method of the present invention makes handling of Bifidobacterium bacteria easier than conventional methods. Bifidobacterium bacteria cultured by the culture method of the present invention are easy to handle in an aerobic environment, and can be provided with a high viability. Furthermore, when the culture method of the present invention is performed in an anaerobic environment, the dissolved oxygen concentration in the medium may be set to a certain concentration or higher. Here, the term "culture environment" refers to the atmosphere surrounding the medium in which the present Bifidobacterium bacteria are cultured.

[0054] When culturing the present Bifidobacterium bacteria in a liquid medium after adjusting the oxygen concentration in the culture environment as described above, the liquid medium may be cultured while stirring. Stirring increases the efficiency of oxygen uptake into the liquid medium in the culture environment, and even if the oxygen concentration in the liquid medium increases, the present Bifidobacterium bacteria can be cultured without problems because they are oxygen-tolerant. Furthermore, culturing while stirring ensures that nutrients are evenly supplied to the liquid medium, improving the growth efficiency of the present Bifidobacterium bacteria.

[0055] After adjusting the oxygen concentration in the culture environment as described above, the present Bifidobacterium bacteria may be seeded on a solid medium and cultured. In the culture method of the present invention, adjusting the oxygen concentration in the culture environment as described above eliminates the need for conventional culture involving the aeration of anaerobic gases such as carbon dioxide. In the culture method of the present invention, handling of Bifidobacterium bacteria is easier than in conventional solid culture.

[0056] The medium for culturing the present Bifidobacterium bacteria is not particularly limited, and media commonly used for culturing Bifidobacterium bacteria can be used. Specifically, sugars such as glucose, galactose, lactose, arabinose, mannose, sucrose, fructose, cellobiose, trehalose, starch, starch hydrolysates, and blackstrap molasses can be used as carbon sources depending on the assimilation potential. Nitrogen sources include ammonium salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate, as well as nitrates. Inorganic salts include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, potassium sulfate, and ferrous sulfate. Organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract can also be used.

[0057] The dissolved oxygen concentration in the medium can be 0.01 to 7 ppm, 0.01 to 3 ppm, or 0.01 to 1 ppm. In the culture method of the present invention, adjusting the dissolved oxygen concentration in the medium as described above eliminates the need for conventional culture involving aeration with anaerobic gases such as carbon dioxide. The culture method of the present invention makes handling of Bifidobacterium bacteria easier than conventional methods. Bifidobacterium bacteria cultured by the culture method of the present invention are easy to handle in an aerobic environment and can therefore be provided with a high viable cell rate. The culture temperature may be 25 to 50°C, and preferably 35 to 42°C.

[0058] <Modified Bifidobacterium> The present invention also relates to a modified Bifidobacterium bacterium having enhanced or conferred oxygen tolerance, which is obtained by introducing any one of the genes selected from (1) to (7) described above. The explanation in the section <Cultivation Method> above applies to the genes (1) to (7).

[0059] As used herein, "enhancing" oxygen tolerance refers to a situation in which oxygen tolerance is increased after the introduction of the gene compared to before. Furthermore, as used herein, "conferring" oxygen tolerance refers to a situation in which oxygen tolerance is acquired by introducing the gene, even though oxygen tolerance was not present before the introduction of the gene. The strength of oxygen tolerance can be quantitatively evaluated by the viable cell rate or CFU when cultured under aerobic conditions.

[0060] The present gene can be introduced into Bifidobacterium bacteria using any known genetic engineering technique without limitation. Examples include the competence method, which utilizes DNA uptake ability, and electroporation, which utilizes high-voltage pulses. Furthermore, when integrating the present gene into the chromosome of Bifidobacterium bacteria, methods utilizing homologous recombination and site-specific integration can be used.

[0061] The modified Bifidobacterium bacterium of the present invention can be cultured under anaerobic conditions, and because the present gene has been introduced, it can also be cultured under aerobic conditions. The modified Bifidobacterium bacterium of the present invention has enhanced or imparted oxygen tolerance in addition to the functions inherent to Bifidobacterium bacteria, and is therefore provided as a Bifidobacterium bacterium that is resistant to oxygen stress and has excellent functionality. The modified Bifidobacterium bacterium of the present invention is easy to handle in an aerobic environment, and can be provided in a state with a high viable cell rate.

[0062] <Activated Bifidobacterium> Another aspect of the present invention relates to activated Bifidobacterium bacteria in which any one gene selected from the above-mentioned (1) to (7) is upregulated. The explanation in the above section <Cultivation Method> applies to the genes (1) to (7). Here, the activated Bifidobacterium bacteria are Bifidobacterium bacteria that have any one gene selected from the above-mentioned (1) to (7) and in which the gene is upregulated. In other words, the activated Bifidobacterium bacteria are Bifidobacterium bacteria that have any one gene selected from the above-mentioned (1) to (7) and in which the expression level of any one gene selected from the above-mentioned (1) to (7) is increased.

[0063] Bifidobacterium bacteria having any one gene selected from (1) to (7) can have the gene upregulated when cultured under aerobic conditions. That is, whether or not a Bifidobacterium bacterium is activated can be confirmed by determining the expression level of a gene product (e.g., mRNA or protein) of any one gene selected from (1) to (7) when cultured under anaerobic and aerobic conditions. Specifically, the expression levels of a Bifidobacterium bacterium having any one gene selected from (1) to (7) when cultured under anaerobic and aerobic conditions are compared. If the expression of the gene is elevated in aerobic culture, the Bifidobacterium bacterium can be confirmed to be the activated Bifidobacterium bacterium of the present invention. Because genes related to oxygen tolerance are upregulated in the activated Bifidobacterium bacterium of the present invention, the activated Bifidobacterium bacterium is provided as a Bifidobacterium bacterium that is resistant to oxygen stress and has excellent functionality. The activated Bifidobacterium bacteria of the present invention can be easily handled in an aerobic environment, and therefore can be provided in a state with a high viable cell rate.

[0064] <Production Method> The present invention relates to a method for producing a composition containing Bifidobacterium bacteria, including the above-mentioned culture method. That is, the production method of the present invention is characterized by including a step of culturing the present Bifidobacterium bacteria under aerobic conditions. The production method of the present invention may include any steps necessary for producing the target composition in addition to the culture step. Since the production method of the present invention uses Bifidobacterium bacteria with excellent oxygen tolerance, not only the culture step but all production steps can be carried out under aerobic conditions. Therefore, the production method of the present invention does not require a step involving aeration with anaerobic gases such as carbon dioxide gas in all production steps, making it easy to handle. The composition obtained by the production method of the present invention is easy to handle in an aerobic environment, and can be provided with a high viability of Bifidobacterium bacteria.

[0065] In the production method of the present invention, the Bifidobacterium bacterium to be cultured under aerobic conditions may be the present Bifidobacterium bacterium, the above-mentioned modified Bifidobacterium bacterium, or an activated Bifidobacterium bacterium, specific embodiments of which are as described above in the sections <Modified Bifidobacterium bacterium> and <Activated Bifidobacterium bacterium>.

[0066] The "composition" produced by the production method of the present invention includes not only final products provided to consumers, such as food compositions and pharmaceutical compositions, but also the present Bifidobacterium culture that has undergone the culture process; additives used to obtain the final product; and intermediate compositions produced in the process of obtaining the final product.

[0067] The present Bifidobacterium bacteria contained in the composition produced by the production method of the present invention may be live or killed. Preferred examples of the final product include food compositions and pharmaceutical compositions. Food compositions and pharmaceutical compositions can be produced by adding live or killed Bifidobacterium bacteria cultured by the above-mentioned culture method to ordinary food or pharmaceutical ingredients. Apart from the addition of the bacteria, they can be produced in the same manner as ordinary foods, beverages, or pharmaceuticals. The bacteria may be added at any stage of the production process. When live Bifidobacterium bacteria are used in the production process, the Bifidobacterium bacteria contained in the composition can be killed by a final sterilization treatment such as heating or pulverization.

[0068] Because the composition produced by the production method of the present invention contains Bifidobacterium bacteria, its ingestion by animals is expected to have an effect of improving the intestinal microflora. The ingestion of the composition may be for therapeutic or non-therapeutic purposes. Here, "non-therapeutic purposes" does not include medical procedures, i.e., therapeutic treatments of the human body. Examples include health promotion and cosmetic procedures. "Improvement" refers to the improvement of a disease, symptom, or condition; the prevention or delay of the worsening of a disease, symptom, or condition; or the reversal, prevention, or delay of the progression of a disease or symptom. "Prevention" refers to the prevention or delay of the onset of a disease or symptom in a subject, or the reduction of the risk of a disease or symptom in a subject.

[0069] When the composition produced by the production method of the present invention is used for non-therapeutic purposes, it can be administered to healthy individuals. A healthy individual may mean an individual who has a healthy intestinal flora and who is not suffering from a disease caused by a disruption of the intestinal flora. Examples of diseases caused by a disruption of the intestinal flora include diarrhea and constipation. When the composition produced by the production method of the present invention is used for non-therapeutic purposes, it can be used to further improve the healthy intestinal flora possessed by healthy individuals. Furthermore, it is possible to prevent diarrhea or constipation in healthy individuals.

[0070] When the composition produced by the production method of the present invention is used for therapeutic purposes, it can be administered to unhealthy individuals. Examples of unhealthy individuals include those suffering from diseases associated with intestinal flora. Examples of such diseases include intestinal diseases, such as small intestinal tumors, gastrointestinal bleeding, intussusception, gastrointestinal stromal tumors, malabsorption syndrome, Behçet's disease, colon cancer, polyps, diverticulitis, acute appendicitis, irritable bowel syndrome, ulcerative colitis, ischemic colon, and Crohn's disease. When the composition produced by the production method of the present invention is used for therapeutic purposes, it can regulate the intestines in unhealthy individuals, improving, preventing, and treating diarrhea and constipation. When the oral composition for intestinal delivery of the present invention is used for therapeutic purposes, it can be used to improve the intestinal flora of unhealthy individuals. The composition produced by the production method of the present invention may be administered to humans or non-human animals (preferably mammals), with humans and pets being preferred, and humans being more preferred. Furthermore, the target population of the present technology is not particularly limited as long as it is anyone desiring a probiotic effect, and examples thereof include infants, children, adults, middle-aged and elderly people, the elderly, healthy individuals, and individuals with poor intestinal environments, etc. Among these, the present technology is preferably used for infants, adults, the elderly, and individuals with poor intestinal environments.

[0071] The composition produced by the production method of the present invention is preferably administered or ingested continuously for at least one week, more preferably at least four weeks, and is desirably ingested daily during the administration or ingestion period.

[0072] The amount of the composition produced by the production method of the present invention to be used is not particularly limited, but is preferably 0.01 to 10,000 mg / kg body weight / day, more preferably 0.01 to 1,000 mg / kg body weight / day, and even more preferably 0.1 to 100 mg / kg body weight / day, in terms of dry weight of the bacterial cells.

[0073] Examples of food compositions include beverages such as infant formula, soft drinks, carbonated drinks, nutritional drinks, fruit juice drinks, and lactic acid bacteria drinks (including concentrated concentrates and powders for preparation of these beverages); frozen desserts such as ice cream, sherbet, and shaved ice; sweets such as candy, chewing gum, candy, chewing gum, chocolate, tablet candy, snacks, biscuits, jelly, jam, cream, and baked goods; dairy products such as processed milk, milk drinks, fermented milk, drinkable yogurt, and butter; bread; enteral nutritional foods, liquid foods such as porridge, baby food, sports drinks; and other functional foods. The food composition may also be a supplement, such as a tablet supplement. Supplements allow the intake of the Bifidobacterium bacteria without being affected by other foods in terms of daily dietary intake and calorie intake. Other commercially available foods include, for example, baby food, sprinkles, and ochazuke nori seaweed. The infant formula includes powdered infant formula and liquid infant formula. Formulated powdered milk is defined in the Ministerial Ordinance on the Compositional Standards of Milk and Dairy Products (Milk Ministerial Ordinance) as "a powder made by processing raw milk, cow's milk, special cow's milk, or foods made from these as ingredients, or using them as the main ingredient, and adding nutrients necessary for infants." Formulated liquid milk is defined in the same ordinance as "a liquid made by processing raw milk, cow's milk, special cow's milk, or foods made from these as ingredients, or using them as the main ingredient, and adding nutrients necessary for infants." Formulated milk is a blend of nutritional ingredients such as various proteins, fats and oils, carbohydrates, minerals, and vitamins, and includes formulas processed into powder or liquid form. Furthermore, the term "formulated milk" further includes "powdered infant formula," "liquid infant formula," and "powdered milk for pregnant and nursing women," which are foods for special dietary uses defined in the Health Promotion Act, as well as powdered infant formula for infants aged 0 to 12 months, follow-up milk for infants aged 6 to 9 months or older and young children (up to 3 years old), powdered infant formula for low birth weight infants weighing less than 2500 g at birth (low birth weight infants), various therapeutic milks used to treat infants with pathological conditions such as cow's milk allergy or lactose intolerance, powdered infant formula, nutritional powder for adults, nutritional powder for the elderly, etc.The food composition can be used for both therapeutic and non-therapeutic purposes, but is preferably used for non-therapeutic purposes in healthy individuals.

[0074] The pharmaceutical composition may be for oral administration. The dosage form of the pharmaceutical composition is not particularly limited. For oral administration, the pharmaceutical composition may be formulated into solid preparations such as powders, granules, tablets, lozenges, and capsules; or liquid preparations such as solutions, syrups, suspensions, and emulsions. For parenteral administration, the pharmaceutical composition may be formulated into suppositories, sprays, inhalants, ointments, patches, and injections. Furthermore, additives commonly used as pharmaceutical carriers, such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, and injectable solvents, may be used in the formulation. These components may be appropriately selected by those skilled in the art depending on the dosage form. The pharmaceutical composition may be used for both therapeutic and non-therapeutic purposes, but is preferably used for therapeutic purposes in healthy subjects.

[0075] When a food composition or pharmaceutical composition is produced by the production method of the present invention, the content of the Bifidobacterium bacteria in the composition is 1 x 10 per day. ５ ~1 x 10 １２ CFU / kg body weight / day, preferably 1 x 10 ７ ~1 x 10 １１ More preferably, 1 x 10 CFU / kg body weight / day ８ ~1 x 10 １０ It is more preferable that the dose is 10 CFU / kg body weight / day. ７ ~10 １４ CFU / day is preferred, 10 ８ ~10 １３ CFU / day is more preferred, 10 ９ ~10 １２ CFU / day is more preferred. If the Bifidobacterium bacteria are sterile, CFU can be substituted with cells.

[0076] The composition produced by the production method of the present invention preferably contains prebiotics, such as indigestible dextrin, inulin, lactulose, galactooligosaccharides, fructooligosaccharides, soybean oligosaccharides, lactoferrin oligosaccharides, xylooligosaccharides, isomaltoligosaccharides, raffinose, coffee bean mannooligosaccharides, gluconic acid, polydextrose, guar gum hydrolysate, alginate, pectin, isomaltodextrin, barley β-glucan, and human milk oligosaccharides (HMOs). Examples of human milk oligosaccharides include 2'-fucosyllactose, 3'-sialyllactose, lacto-N-tetraose 3-difucosyllactose, 3-fucosyllactose, 3-fucosyl-3'-sialyllactose, lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-fucopentaose III, lacto-N-fucopentaose V, lacto-N-difucosylhexaose I, lacto-N-difucosylhexaose II, lacto-N-sialylpentaose, LSTa, LSTb, and LSTc.

[0077] One embodiment of the production method of the present invention includes a step of drying a bacterial liquid containing the present Bifidobacterium bacteria that has been cultured by the above-mentioned culture method. According to this embodiment, a powder of the present Bifidobacterium bacteria can be obtained. The production method of the present invention uses Bifidobacterium bacteria that have excellent oxygen tolerance, so the drying step can be carried out under aerobic conditions. The production method of the present invention can provide a composition that has a high viability rate of Bifidobacterium bacteria even after the drying step.

[0078] The drying method is not particularly limited, but examples thereof include freeze drying and spray drying.

[0079] In the bacterial powder, the cells of the Bifidobacterium genus may be crushed, and the crushed material may be obtained by crushing live cells or dead cells, or may be crushed and then subjected to heating, freeze-drying, etc. Of these, from the viewpoint of increasing the viable cell rate, it is preferable to subject a culture of the Bifidobacterium genus to freeze-drying, and from the viewpoint of increasing production efficiency, it is preferable to subject the culture to spray-drying.

[0080] In one embodiment, a food composition is produced by adding the bacterial powder obtained by the drying process described above to a raw material composition. The food composition that can be produced in this embodiment is as described above. The raw material composition can be selected appropriately depending on the food composition that is the target of production. In one embodiment, the bacterial powder itself is added to the raw material composition, or a solution obtained by dissolving the bacterial powder in water or the like is added to the raw material composition. In the production method of the present invention, a food composition is produced by adding the bacterial powder obtained by the drying process described above to the raw material composition, so that a food composition with a high viable cell rate of Bifidobacterium bacteria can be provided.

[0081] Furthermore, food compositions may be produced through a fermentation process using the added Bifidobacterium bacteria, such as lactic acid bacteria drinks and fermented milk.

[0082] In one embodiment, the method includes a step of adding a starter culture containing the present Bifidobacterium bacteria cultured by the above-mentioned culture method to a raw material composition and fermenting the mixture. This embodiment is effective, for example, when producing a lactic acid bacteria beverage or fermented milk.

[0083] The starter culture may contain other bacteria in addition to the Bifidobacterium bacteria of the present invention. Examples of such other bacteria include lactic acid bacteria, such as Lactococcus lactis, Streptococcus thermophilus, and Lactobacillus delbrueckii. While the subspecies of Lactococcus lactis are not particularly limited, examples of Lactococcus lactis include Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. Examples of Lactobacillus delbrueckii include Lactobacillus delbrueckii subsp. bulgaricus.

[0084] The composition produced by the production method of the present invention may be provided as a product housed in a packaging container that is an aerobic environment. Because the Bifidobacterium bacterium of the present invention is easy to handle in an aerobic environment, a high viable cell rate can be maintained even when the composition is provided in a packaging container that is an aerobic environment.

[0085] <Screening Method> The present invention also relates to a screening method for oxygen-tolerant Bifidobacterium bacteria, which comprises a step of selecting Bifidobacterium bacteria using any one gene selected from the above-mentioned (1) to (7) as an indicator.

[0086] As described above, Bifidobacterium bacteria carrying the present gene have oxygen tolerance, allowing them to be cultured under aerobic conditions. In other words, Bifidobacterium bacteria carrying the present gene can be determined to have oxygen tolerance.

[0087] The screening method of the present invention may be an embodiment in which a test Bifidobacterium bacterium is determined to have oxygen tolerance when it has either a gene consisting of the base sequence represented by SEQ ID NO: 1 or a gene consisting of the base sequence represented by SEQ ID NO: 2, or an embodiment in which a test Bifidobacterium is determined to have oxygen tolerance when it has both of these genes.

[0088] The screening method of the present invention may be an embodiment in which a test Bifidobacterium bacterium is determined to have oxygen tolerance when it has either a gene having 90% or more identity to the base sequence represented by SEQ ID NO: 1 or a gene having 90% or more identity to the base sequence represented by SEQ ID NO: 2, or an embodiment in which a test Bifidobacterium is determined to have oxygen tolerance when it has both of these genes.

[0089] The screening method of the present invention may be an embodiment in which a test Bifidobacterium bacterium is determined to have oxygen tolerance when it has either a gene consisting of a base sequence in which one to several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 1, or a gene consisting of a base sequence in which one to several bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 2, or an embodiment in which a test Bifidobacterium bacterium is determined to have oxygen tolerance when it has both of these genes.

[0090] The screening method of the present invention may be an embodiment in which a test Bifidobacterium bacterium is determined to have oxygen tolerance when it has either: a gene consisting of a DNA base sequence that hybridizes under stringent conditions with DNA consisting of a complementary sequence to the base sequence shown in SEQ ID NO: 1; or a gene consisting of a DNA base sequence that hybridizes under stringent conditions with DNA consisting of a complementary sequence to the base sequence shown in SEQ ID NO: 2; or an embodiment in which a test Bifidobacterium is determined to have oxygen tolerance when it has both of these genes.

[0091] The screening method of the present invention may be an embodiment in which a test Bifidobacterium bacterium is determined to have oxygen tolerance when it has either a gene consisting of a degenerate isomer of the base sequence represented by SEQ ID NO: 1 or a gene consisting of a degenerate isomer of the base sequence represented by SEQ ID NO: 2, or an embodiment in which a test Bifidobacterium is determined to have oxygen tolerance when it has both of these genes.

[0092] The screening method of the present invention may be an embodiment in which a test Bifidobacterium bacterium is determined to have oxygen tolerance when it has either a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 4, or an embodiment in which a test Bifidobacterium is determined to have oxygen tolerance when it has both of these genes.

[0093] The screening method of the present invention may be an embodiment in which a test subject Bifidobacterium bacterium is determined to have oxygen tolerance when it has either a gene encoding a protein consisting of an amino acid sequence in which one to several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 3, or a gene encoding a protein consisting of an amino acid sequence in which one to several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 4, or an embodiment in which a test subject Bifidobacterium bacterium is determined to have oxygen tolerance when it has both of these genes.

[0094] The above-mentioned explanation in the section <Culture Method> applies to the above-mentioned genes (1) to (7) that serve as indicators for the screening method of the present invention.

[0095] For the screening method of the present invention, the method for determining the presence or absence of the present gene is not particularly limited. The presence or absence of the present gene may be determined using genomic DNA as a target, or the presence or absence of the present gene may be indirectly determined by measuring the presence or absence of mRNA, which is its transcription product, or protein, which is its translation product.

[0096] For example, primers that specifically hybridize to the base sequence region of the present gene may be designed, and the presence or absence of the present gene may be determined by PCR using the genome of the test Bifidobacterium bacterium as a template. Alternatively, the presence or absence of the present gene may be determined indirectly by extracting mRNA from the test Bifidobacterium bacterium and using RT-PCR or an array that can specifically detect the transcription product of the present gene. Furthermore, the presence or absence of the present gene may be determined indirectly by techniques such as Western blotting using an antibody against the protein encoded by the present gene. The screening method of the present invention enables easy screening of Bifidobacterium bacteria with excellent oxygen tolerance using the above-mentioned genes (1) to (7) as indicators.

[0097] <Starter culture composition> The present invention also relates to a starter culture composition comprising a Bifidobacterium bacterium having any one of the genes selected from (1) to (7) above. The explanation in the section <Cultivation method> above applies to the genes (1) to (7). The starter culture composition of the present invention can be obtained by inoculating and culturing the Bifidobacterium bacterium in a medium.

[0098] In the starter culture composition of the present invention, a modified Bifidobacterium bacterium can be used as the Bifidobacterium bacterium. That is, another embodiment of the starter culture composition of the present invention includes a modified Bifidobacterium bacterium having enhanced or conferred oxygen tolerance, which is obtained by introducing any one gene selected from the above-mentioned (1) to (7). With regard to the modified Bifidobacterium bacterium, the explanation in the above section <Modified Bifidobacterium bacterium> is applicable.

[0099] In the starter culture composition of the present invention, activated Bifidobacterium bacteria can be used as the Bifidobacterium bacteria. That is, another embodiment of the starter culture composition of the present invention comprises activated Bifidobacterium bacteria in which any one gene selected from the above-mentioned (1) to (7) is upregulated. With regard to the activated Bifidobacterium bacteria, the explanation in the above section <Activated Bifidobacterium bacteria> is applicable.

[0100] The starter culture composition of the present invention can be in the form of a fermented milk starter composition, which can be obtained by inoculating and culturing the present Bifidobacterium bacterium in a milk medium consisting of whole milk, skim milk, or reconstituted milk, or in a synthetic or semi-synthetic medium containing lactose, glucose, or the like as a main component.

[0101] When the starter culture composition of the present invention is used to produce fermented compositions such as fermented milks such as drinkable yogurt and acidic milk drinks, it is possible to obtain a product with a high viable cell count of the present Bifidobacterium bacteria.

[0102] The starter culture composition of the present invention can be embodied as containing lactic acid bacteria in addition to the Bifidobacterium bacteria of the present invention. More specific details of this embodiment will be described in detail in the section "Composition" below.

[0103] <Composition> The present invention also relates to a composition comprising a Bifidobacterium bacterium having any one of the genes (1) to (7) above, and one or more lactic acid bacteria selected from Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus delbrueckii. The explanation in the section <Cultivation Method> above applies to the genes (1) to (7).

[0104] In the composition invention, a modified Bifidobacterium bacterium can be used as the Bifidobacterium bacterium. That is, another embodiment of the composition invention comprises a modified Bifidobacterium bacterium having enhanced or conferred oxygen tolerance, which has been introduced with any one gene selected from the above-mentioned (1) to (7), and one or more lactic acid bacteria selected from Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus delbrueckii. With regard to the modified Bifidobacterium bacterium, the explanation in the above section <Modified Bifidobacterium bacterium> is applicable.

[0105] In the composition invention, activated Bifidobacterium bacteria can be used as the Bifidobacterium bacteria. That is, another embodiment of the composition invention comprises an activated Bifidobacterium bacterium in which any one of the genes selected from (1) to (7) above is upregulated, and one or more lactic acid bacteria selected from Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus delbrueckii. The explanation in the above section <Activated Bifidobacterium bacteria> applies to the activated Bifidobacterium bacteria.

[0106] The present Bifidobacterium bacteria and the above-mentioned lactic acid bacteria have oxygen tolerance, allowing them to be cultured even under aerobic conditions. In other words, the composition of the present invention can be said to be a mixture of bifidobacteria and lactic acid bacteria that can be cultured even under aerobic conditions. Due to these properties, the composition of the present invention is useful as a starter culture.

[0107] By using the composition of the present invention as a starter culture, a fermentation composition having a high viability of the present Bifidobacterium bacteria and lactic acid bacteria can be provided.

[0108] The composition of the present invention can be in the form of a fermented milk starter composition, which can be obtained by inoculating and culturing the present Bifidobacterium bacterium in a milk medium made from whole milk, skim milk, reconstituted milk, or the like, or in a synthetic or semi-synthetic medium containing lactose, glucose, or the like as a main component.

[0109] When the composition of the present invention is used as a fermented milk starter to produce fermented compositions such as fermented milk, such as drinkable yogurt and acidic milk drinks, it is possible to obtain products with high viable counts of the present Bifidobacterium bacteria and lactic acid bacteria.

[0110] <Test Example> (1) Bifidobacterium bacteria used In this test example, the following eight strains of Bifidobacterium bacteria were used: MCC02042 (Example 1): Bifidobacterium longum subsp. infantis specified in NITE BP-03068; MCC10110 (Example 2): Bifidobacterium longum subsp. infantis. Strains stored at the Research and Information Center of Morinaga Milk Industry Co., Ltd. (5-1-83 Higashihara, Zama City, Kanagawa Prefecture, Postal Code 252-8583) JCM1210: Bifidobacterium longum subsp. infantis identified by JCM number JCM1210; JCM1222: Bifidobacterium longum subsp. infantis identified by JCM number JCM1222; JCM1260: Bifidobacterium longum subsp. infantis identified by JCM number JCM1260; JCM11344: Bifidobacterium longum subsp. infantis identified by JCM number JCM11344; JCM11345: Bifidobacterium longum subsp. infantis identified by JCM number JCM11345; JCM11347: Bifidobacterium longum subsp. infantis identified by JCM number JCM11347;

[0111] (2) Oxygen Tolerance Test Each strain (MCC2042, MCC10110, JCM1210, JCM1222, JCM1260, JCM11344, JCM11345, JCM11347) was cultured in MRS medium (manufactured by BD Difco) at 37 ° C. under anaerobic conditions for 16 hours. 100 μL of the cultured bacterial solution was spread on BL agar medium (manufactured by Eiken Chemical Co., Ltd.) and cultured at 37 ° C. under aerobic conditions for 48 hours. The presence or absence of colony formation on BL agar medium was evaluated. As a result, only MCC2042 and MCC10110 formed colonies, while the other strains did not. Based on the above results, MCC2042 and MCC10110 were determined to be oxygen-tolerant strains.

[0112] (3) Draft Genome Sequence Each strain (JCM1210, JCM1222, JCM1260, JCM11344, JCM11345, JCM11347) was cultured in MRS medium at 37 °C under anaerobic conditions for 16 hours. The culture was centrifuged to obtain a bacterial pellet. DNA extraction was performed using a DNeasy Blood & Tissue Kit (QIAGEN). A genomic library was prepared using 1 ng of extracted DNA using a Nextera XT DNA Sample Preparation Kit (Illumina). After amplification by PCR (Thermo Fisher Scientific, Veriti200) and cleanup, the fragment size distribution of the tagged DNA was analyzed using an Agilent 2100 Bioanalyzer and a High Sensitivity DNA Analysis Kit (Agilent).

[0113] The library was sequenced using the MiSeq Personal Sequencing System and MiSeq Reagent Kit v2 (500 cycles). The resulting sequence data was trimmed and assembled using CLC Genomics Workbench (QIAGEN). The settings were default, but contigs shorter than 2000 bp were removed.

[0114] (4) Complete Genome Sequencing Each strain (MCC2042, MCC10110) was cultured in MRS medium at 37°C under anaerobic conditions for 16 hours. The culture was centrifuged to obtain a bacterial pellet. DNA was extracted using a NucleoBond high-molecular-weight (HMW) DNA kit (Machrei-Nagel). Genomic libraries were prepared using an SMRTbell Express Template Prep Kit (Pacific Biosciences). Size distribution was confirmed by pulsed-field electrophoresis, and size selection was performed using BluePippi (Sage Science). Sequence analysis was performed using the long-read sequencers PacBio RSII and SMRT Cell 8 Pac V3. The obtained sequence data was assembled using canu. Default settings were used.

[0115] (5) Comparative Genome Analysis Using the acquired complete genome and draft genome fasta files as input, annotation was performed using bakta to obtain faa files. Using all acquired faa files as input, an ortholog table was obtained using sonic paranoid2 (Non-Patent Document 1). The specified parameters were --mmseqs 7.5, --go. From the acquired ortholog table, ortholog groups present only in MCC2042 and MCC10110 and not present in other strains were extracted, and a list of amino acid sequences of MCC2042 genes included in the ortholog groups was obtained. The EC numbers (Enzyme Commission numbers) of these amino acid sequences were predicted using CLEAN (Non-Patent Document 2).

[0116] As shown in (2) above, MCC2042 and MCC10110 were able to form colonies under aerobic conditions. These two strains were thought to have a higher ability to decompose oxygen than other strains. In other words, it was presumed that the gene responsible for the oxygen tolerance exhibited by these two strains was a gene encoding an oxidoreductase (a group of enzymes whose first EC number is 1; hereinafter, also referred to as "enzymes with EC numbers in the 1 range").

[0117] Based on this assumption, the list of amino acid sequences obtained by the comparative genome analysis described above and the predicted EC numbers were checked. As a result, a gene consisting of the base sequence represented by SEQ ID NO: 1 or 2 was identified as a gene not possessed by other strains but possessed only by MCC2042 and MCC10110, and as a gene encoding the amino acid sequence of an enzyme group (oxidoreductase) with an EC number in the 1 range.

[0118] Furthermore, among the genes that are not possessed by other strains but are possessed only by MCC2042 and MCC10110, genes other than the genes consisting of the base sequences represented by SEQ ID NO: 1 or 2 were examined in detail based on the estimated EC numbers, but it was not possible to rationally deduce the possibility that they may be involved in oxygen tolerance.

[0119] From the above, it was concluded that the gene responsible for oxygen tolerance, which was confirmed only in MCC2042 and MCC10110 in the test (2) above, is a gene consisting of the base sequence shown in SEQ ID NO: 1 or 2.

[0120] Preparation Example 1: Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068) was added to 3 mL of MRS liquid medium and cultured under aerobic conditions at 37°C for 24 hours. The culture was then concentrated and freeze-dried to obtain a freeze-dried bacterial powder (bacterial powder). The bacterial powder, whey protein concentrate (WPC), and prebiotics (2'-FL and LNT) were uniformly mixed to obtain a composition. 20 g of the composition was dissolved in 200 g of water to obtain a composition containing Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068).

[0121] <Production Example 2> Skim milk powder, cream, and milk protein were mixed and dissolved to prepare 50 L of a milk raw material consisting of 3.0% (w / w) milk fat and 12.0% (w / w) non-fat milk solids, which was then heated to 70°C, homogenized under a pressure of 15 MPa, sterilized at 90°C for 10 minutes, and cooled to 40°C. 500 mL of a culture of Lactococcus lactis subsp. lactis and 1.5 x 10 frozen cells of Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068) (manufactured by Morinaga Milk Industry Co., Ltd.) were added to this sterilized milk raw material. １４ The culture medium was inoculated with 0.002% yogurt starter containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and cultured for 4 hours at 37° C. to obtain fermented milk. 100 g of this fermented milk was filled into a 120 mL paper container (manufactured by Shingi Co., Ltd., 71 mm diameter, 56 mm height), sealed with an aluminum foil lid, and cooled to 10° C. to obtain a plain fermented milk product.

[0122] <Production Example 3> Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068) was added to 3 mL of MRS liquid medium and cultured under aerobic conditions at 37°C for 24 hours. The culture solution was then concentrated and freeze-dried to obtain a freeze-dried powder of the bacteria (bacterial powder). 10 kg of desalted milk whey protein powder (Mirai Co., Ltd.), 6 kg of milk casein powder (Fonterra), 48 kg of lactose (Mirai Co., Ltd.), 920 g of mineral mixture (Tomita Pharmaceutical Co., Ltd.), 32 g of vitamin mixture (Tanabe Pharmaceutical Co., Ltd.), 500 g of lactulose (Morinaga Milk Industry Co., Ltd.), 500 g of raffinose (Nippon Beet Sugar Co., Ltd.), and 900 g of galactooligosaccharide liquid sugar (Yakult Pharmaceutical Co., Ltd.) were dissolved in 300 kg of warm water, further heated and dissolved at 90°C for 10 minutes, and 28 kg of modified fat (Taiyo Yushi Co., Ltd.) was added and homogenized. The mixture was then sterilized, concentrated, and spray-dried to prepare approximately 95 kg of modified milk powder. To this, freeze-dried powder (1.8 × 10) of the above-mentioned Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068) dispersed in starch was added. １１ cfu / g) to prepare approximately 95 kg of bifidobacteria-oligosaccharide-containing infant formula. The resulting infant formula was dissolved in water to prepare a milk preparation with a total solids concentration of 14% (w / v), which is the standard milk preparation concentration. The number of bifidobacteria in the milk preparation was 2.7 x 10 ９ cfu / 100 mL can be obtained.

[0123] <Reference Example> Using a temperature-sensitive plasmid developed for genetic modification of bifidobacteria, deletion of SEQ ID NOs: 1 and 2, which are genes responsible for oxygen tolerance, is carried out based on Non-Patent Document 3. 500-1000 bases of the upstream and downstream base sequences of the genes of SEQ ID NOs: 1 and 2 are selected and amplified by PCR. Primers with 15-25 base overlap sequences added to the 5' ends are used for binding between the temperature-sensitive plasmid and the amplified sequence by In-Fusion reaction, as well as for binding between the upstream and downstream amplified sequences. The temperature-sensitive plasmid is linearized by restriction enzyme treatment. The linearized plasmid and the amplified base sequence are mixed and ligated using In-Fusion Snap Assembly Master Mix (TaKaRa Bio). The ligated plasmid is transformed into DH5α (TaKaRa Bio) and amplified. The amplified plasmid is extracted and used to transform B. The plasmid is introduced into B. infantis MCC02042 by electroporation. After electroporation, B. infantis MCC02042 is cultured on GAM agar medium containing spectinomycin at 30°C. To induce single crossover, B. infantis MCC02042 transformed with the plasmid is cultured on the Gifu anaerobic medium (GAM) agar medium containing spectinomycin at 39°C to 42°C. The formed colonies are scraped and cultured for 1-2 days at 30°C in the de Man, Rogosa, and Sharpe (MRS) medium supplemented with 0.05% cysteine. This bacterial solution is plated on MRS agar medium and cultured at 37°C for 1 day. The colonies are replicated and further cultured at 37°C. After culturing, colonies that do not grow are picked and confirmed by PCR for the presence or absence of deletion of the oxygen tolerance genes SEQ ID NOS: 1 and 2. If the strain is defective, the defective strain is cultured in BL medium at 37°C under aerobic conditions for approximately 72 hours, and the difference in growth compared with the wild-type strain (MCC2042) is compared to determine whether SEQ ID NOS: 1 and 2 are the genes responsible for oxygen tolerance.

[0124] The present invention can be applied to the production technology of foods or pharmaceuticals containing oxygen-tolerant Bifidobacterium bacteria.

Claims

1. A method for culturing Bifidobacterium bacteria having any one gene selected from the following (1) to (7), under aerobic conditions: (1) a gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (2) a gene having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 or 2. (3) a gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2, in which one to several nucleotides have been deleted, substituted, or added. (4) a gene consisting of a DNA nucleotide sequence that hybridizes under stringent conditions with DNA consisting of the complementary sequence of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (5) a gene consisting of a degenerate isomer of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (6) a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4. (7) a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4, in which one to several amino acids have been deleted, substituted, or added.

2. The culture method according to claim 1, wherein the Bifidobacterium bacterium is Bifidobacterium longum.

3. The culture method according to claim 1, wherein the Bifidobacterium bacterium is Bifidobacterium longum subsp. infantis.

4. The culture method according to claim 1, wherein the Bifidobacterium bacterium is Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068).

5. A method for producing a composition containing Bifidobacterium bacteria, comprising the culture method according to any one of claims 1 to 4.

6. The method according to claim 5, further comprising a step of drying a bacterial liquid containing the Bifidobacterium bacteria cultured by the culture method, wherein the composition is a bacterial powder obtained by the drying or a food composition to which the bacterial powder has been added.

7. The production method according to claim 5, comprising the step of adding a starter culture containing the Bifidobacterium bacteria cultured by the culture method to a raw material composition and fermenting the mixture.

8. A modified Bifidobacterium bacterium having enhanced or conferred oxygen tolerance, which is obtained by introducing any one gene selected from the following (1) to (7): (1) A gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (2) A gene having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 or 2. (3) A gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2, in which one to several nucleotides have been deleted, substituted, or added. (4) A gene consisting of a DNA nucleotide sequence that hybridizes under stringent conditions with DNA consisting of the complementary sequence of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (5) A gene consisting of a degenerate isomer of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (6) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4. (7) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4, in which one to several amino acids have been deleted, substituted, or added.

9. A method for screening for oxygen-tolerant Bifidobacterium bacteria, comprising the step of selecting Bifidobacterium bacteria using any one gene selected from the following (1) to (7) as an index: (1) A gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (2) A gene having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 or 2. (3) A gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2, in which one to several bases are deleted, substituted, or added. (4) A gene consisting of a DNA nucleotide sequence that hybridizes under stringent conditions with DNA consisting of the complementary sequence of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (5) A gene consisting of a degenerate isomer of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (6) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4. (7) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4, in which one to several amino acids are deleted, substituted, or added.

10. A starter culture composition comprising a Bifidobacterium bacterium having any one gene selected from the following (1) to (7): (1) A gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (2) A gene having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 or 2. (3) A gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2, in which one to several nucleotides are deleted, substituted, or added. (4) A gene consisting of a DNA nucleotide sequence that hybridizes under stringent conditions with DNA consisting of the complementary sequence of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (5) A gene consisting of a degenerate isomer of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (6) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4. (7) A gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4, in which one to several amino acids are deleted, substituted, or added.

11. A Bifidobacterium bacterium having any one gene selected from the following (1) to (7): (1) a gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (2) a gene having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 1 or 2. (3) a gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 2, in which one to several bases are deleted, substituted, or added. (4) a gene consisting of a DNA nucleotide sequence that hybridizes under stringent conditions with DNA consisting of a complementary sequence to the nucleotide sequence represented by SEQ ID NO: 1 or 2. (5) a gene consisting of a degenerate isomer of the nucleotide sequence represented by SEQ ID NO: 1 or 2. (6) a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4. (7) a gene encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 4, in which one to several amino acids are deleted, substituted, or added. A composition comprising one or more lactic acid bacteria selected from Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus delbrueckii.