Bifidobacterium bacteria having oxygen tolerance, culture method, screening method, and composition containing bifidobacterium bacteria having oxygen tolerance and manufacturing method thereof

Abstract

The objective of this invention is to provide: aerobic-tolerant Bifidobacterium spp. capable of being cultured under aerobic conditions, a method for culturing the same and a method for screening it, and a composition comprising aerobic-tolerant Bifidobacterium spp. and a method for manufacturing the same. The present invention that solves this problem is a method for culturing Bifidobacterium bacteria, comprising the step of culturing Bifidobacterium bacteria having any one of the following (1) to (7) under aerobic conditions: (1) a gene consisting of a base sequence shown in sequence number 1 or 2; (2) a gene having more than 90% identity with a base sequence shown in sequence number 1 or 2; (3) a gene consisting of a base sequence in which 1 to several bases are missing, substituted, or added to a base sequence shown in sequence number 1 or 2; (4) a gene consisting of a base sequence of DNA that can hybridize under strict conditions with a DNA consisting of a complementary sequence of a base sequence shown in sequence number 1 or 2; (5) a gene consisting of a degenerate isomer of a base sequence shown in sequence number 1 or 2; (6) a gene encoding a protein consisting of an amino acid sequence shown in sequence number 3 or 4; (7) a gene encoding a protein consisting of an amino acid sequence in which 1 to several amino acids are missing, substituted, or added to an amino acid sequence shown in sequence number 3 or 4.

Description

Technical Field

[0001] This invention relates to oxygen-tolerant Bifidobacterium bacteria, their culture and screening methods, compositions comprising oxygen-tolerant Bifidobacterium bacteria, and methods for manufacturing the same. Background Technology

[0002] Bifidobacteria, a type of gut microbiota, have been found to have numerous health benefits for humans and are widely used in food, beverages, and pharmaceuticals. Many drugs and foods utilize Bifidobacteria, especially in fermented milk (yogurt) and other dairy products. However, Bifidobacteria are obligate anaerobic bacteria that require anaerobic conditions to survive, making them difficult to manipulate in aerobic environments.

[0003] In light of this issue, the following solution was proposed: to isolate a strain of Bifidobacterium brevesus (Bifidobacterium breve SBR3212 (Micro-engineering Research Institute of Bacteria No. 11915)) that exhibits excellent aerobic growth from the feces of healthy breastfed infants and to use it as a starter for fermented milk (Patent Document 1).

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 04-320642

[0007] Non-patent literature

[0008] Non-patent document 1: 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 / 2023.05.14.540736

[0009] Non-patent literature 2: Yu T, Cui H, Li JC, Luo Y, Jiang G, Zhao H., "Enzymefunction prediction using contrastive learning.", Science. 2023 Mar 31;379(6639):1358-1363.doi:10.1126 / science.adf2465.Epub 2023 Mar 30.,PMID:36996195

[0010] Non-patent literature 3: 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. Summary of the Invention

[0011] The problem the invention aims to solve

[0012] Patent document 1 discloses aerobic-tolerant Bifidobacterium bacteria that can be cultured under aerobic conditions. However, it is unclear under what conditions these Bifidobacterium bacteria, previously known as obligate anaerobes, would exhibit aerobic tolerance.

[0013] In view of such problems, the object of the present invention is to provide: aerobic Bifidobacterium bacteria, a method for culturing them and a method for screening them, and a composition comprising aerobic Bifidobacterium bacteria and a method for manufacturing them.

[0014] Solution for solving the problem

[0015] Through repeated and in-depth research, the inventors have finally determined that Bifidobacteria with genes consisting of the base sequence shown in sequence number 1 or 2 possess oxygen resistance, thus completing this invention. The invention that solves the above-mentioned problems is described below.

[0016] [1] A method for culturing Bifidobacterium bacteria, comprising the step of culturing Bifidobacterium bacteria having any one of the genes selected from (1) to (7) under aerobic conditions:

[0017] (1) A gene consisting of the base sequence indicated by sequence number 1 or 2.

[0018] (2) Genes that have more than 90% identity with the base sequence shown in sequence number 1 or 2.

[0019] (3) A gene consisting of a base sequence in which one or more bases are missing, substituted or added in the base sequence shown in sequence number 1 or 2.

[0020] (4) A gene consisting of the base sequence of a DNA that can hybridize under strict conditions with a DNA consisting of a complementary sequence of the base sequence shown in sequence number 1 or 2.

[0021] (5) Genes consisting of degenerate isomers of the base sequence shown in sequence number 1 or 2.

[0022] (6) Genes that encode proteins consisting of the amino acid sequence indicated by sequence number 3 or 4.

[0023] (7) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted or added in the amino acid sequence shown in sequence number 3 or 4.

[0024] [2] According to the culture method described in [1], the aforementioned Bifidobacterium species is Bifidobacterium longum.

[0025] [3] According to the culture method described in [1], the aforementioned Bifidobacterium species is Bifidobacterium longum infantis subspecies.

[0026] [4] According to the culture method described in [1], the aforementioned Bifidobacterium species is Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068).

[0027] [5] A method for manufacturing a composition comprising Bifidobacterium bacteria, comprising the culture method described in any one of [1] to [4].

[0028] [6] The manufacturing method according to [5] includes the following steps: drying the bacterial solution containing the aforementioned Bifidobacterium bacteria obtained by the aforementioned culture method.

[0029] The aforementioned composition is a bacterial powder obtained through the aforementioned drying process or a food composition in which the aforementioned bacterial powder has been added.

[0030] [7] The manufacturing method according to [5] includes the following steps: adding a starter culture containing Bifidobacterium bacteria to the raw material composition and fermenting it, wherein the Bifidobacterium bacteria are obtained by culturing based on the aforementioned culture method.

[0031] [8] A modified Bifidobacterium bacterium that has been enhanced or conferred with oxygen resistance, having been introduced with any of the genes selected from (1) to (7) above.

[0032] [9] A method for screening Bifidobacterium bacteria with oxygen tolerance, comprising a step of selecting Bifidobacterium bacteria using any one of the genes selected from (1) to (7) above as an indicator.

[0033]

[10] A composition for starter culture comprising a Bifidobacterium bacterium having any one of the genes selected from (1) to (7) above.

[0034]

[11] A composition comprising:

[0035] Bifidobacteria possessing any one of the genes selected from (1) to (7) above; and

[0036] It is selected from one or more lactic acid bacteria from Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus delbrueckii.

[0037] The effects of the invention

[0038] According to the present invention, the following can be provided: oxygen-resistant Bifidobacterium bacteria, their culture and screening methods, and compositions comprising the aforementioned Bifidobacterium bacteria and their manufacturing methods. Detailed Implementation

[0039] Preferred embodiments of the present invention will be described. However, the present invention is not limited to the following preferred embodiments, and modifications can be freely made within the scope of the present invention. In this specification, unless otherwise specified, percentages are expressed based on mass.

[0040] <Cultivation Methods>

[0041] The cultivation method of the present invention is characterized by a step of culturing Bifidobacterium bacteria having any one of the genes selected from (1) to (7) below under aerobic conditions.

[0042] It should be noted that (1) the gene consisting of the base sequence shown in sequence number 1 or 2 was discovered through comparative genomic analysis of the experimental examples described later.

[0043] (1) A gene consisting of the base sequence indicated by sequence number 1 or 2.

[0044] (2) Genes that have more than 90% identity with the base sequence shown in sequence number 1 or 2.

[0045] (3) A gene consisting of a base sequence in which one or more bases are missing, substituted or added in the base sequence shown in sequence number 1 or 2.

[0046] (4) A gene consisting of the base sequence of a DNA that can hybridize under strict conditions with a DNA consisting of a complementary sequence of the base sequence shown in sequence number 1 or 2.

[0047] (5) Genes consisting of degenerate isomers of the base sequence shown in sequence number 1 or 2.

[0048] (6) Genes that encode proteins consisting of the amino acid sequence indicated by sequence number 3 or 4.

[0049] (7) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted or added in the amino acid sequence shown in sequence number 3 or 4.

[0050] It should be noted that the base sequences shown by sequence number 1 and sequence number 2, and the amino acid sequences shown by sequence number 3 and 4 are respectively shown in the following table.

[0051] [Table 1]

[0052]

[0053] Bifidobacteria with any of the genes selected from (1) to (7) are oxygen-resistant and can therefore be cultured under aerobic conditions.

[0054] For ease of explanation, the term "any gene selected from (1) to (7)" will sometimes be referred to as "the gene of the present invention". In addition, a Bifidobacterium bacterium having any gene selected from (1) to (7) will sometimes be referred to as "the Bifidobacterium bacterium of the present invention".

[0055] The Bifidobacterium bacteria of this invention can be obtained from animals, including humans, and from the environment. Furthermore, the Bifidobacterium bacteria of this invention can also be artificially manufactured by introducing the genes of this invention through genetic engineering methods.

[0056] The Bifidobacterium bacteria of the present invention can be bacteria obtained through straining with the same genetic background. Furthermore, the Bifidobacterium bacteria of the present invention can also be a common bacterial population that, although having different genetic backgrounds, possesses the genes of the present invention.

[0057] The *Bifidobacterium* genus used in this invention is not particularly limited, but may include, for example, *Bifidobacterium longum*, *Bifidobacterium breve*, *Bifidobacterium bifidum*, *Bifidobacterium adolescentis*, *Bifidobacterium animalis*, *Bifidobacterium pseudolongum*, *Bifidobacterium reuteri*, *Bifidobacterium catenulatum*, *Bifidobacterium pseudocatenulatum*, and *Bifidobacterium lactis*.

[0058] In a preferred embodiment of the present invention, the Bifidobacterium species is Bifidobacterium longum.

[0059] More specifically, the preferred Bifidobacterium species of the present invention is Bifidobacterium longum subsp. infantis.

[0060] Furthermore, more specifically, the Bifidobacterium species of the present invention are preferably Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068).

[0061] In one embodiment, the Bifidobacterium species of the present invention do not include Bifidobacterium breve SBR3212 (Microbial Research Institute of Bacteria No. 11915).

[0062] [Gene]

[0063] (1) Genes consisting of the base sequence shown in sequence number 1 or 2

[0064] The gene consisting of the base sequence shown in sequence number 1 is the gene encoding SDR (NADH oxidoreductase).

[0065] The gene consisting of the base sequence shown in sequence number 2 is a gene that encodes a transcriptional regulatory factor.

[0066] The two genes mentioned above are common to strains of Bifidobacterium bacteria that are aerobic and capable of being cultured under aerobic conditions, and are concluded to be related to aerobic tolerance. Therefore, Bifidobacterium bacteria possessing genes consisting of the base sequences shown in sequence number 1 or 2 have aerobic tolerance and can be cultured under aerobic conditions.

[0067] The Bifidobacterium bacteria of the present invention can have a morphology having any one of the following genes, or a morphology having both of the following genes:

[0068] A gene consisting of the base sequence shown in sequence number 1; and

[0069] A gene consisting of the base sequence shown in sequence number 2.

[0070] (2) Genes that have more than 90% identity with the base sequence shown in sequence number 1 or 2

[0071] The Bifidobacterium bacteria of the present invention can have a morphology having any one of the following genes, or a morphology having both of the following genes:

[0072] Genes that share more than 90% identity with the base sequence shown in sequence number 1; and

[0073] Genes that share more than 90% identity with the base sequence shown in sequence number 2.

[0074] In the aforementioned (2) genes, the aforementioned identity is preferably 93% or more, more preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more.

[0075] The gene in (2) above is preferably a gene that has more than 90% identity with the base sequence shown in sequence number 1 or 2, and has the function of enhancing or conferring oxygen resistance when the same gene is introduced into Bifidobacterium bacteria.

[0076] (3) Genes consisting of base sequences in which one or more bases are deleted, substituted, or added in the base sequence shown in sequence number 1 or 2.

[0077] The Bifidobacterium bacteria of the present invention can have a morphology having any one of the following genes, or a morphology having both of the following genes:

[0078] Genes consisting of base sequences with deletions, substitutions, or additions of one to several bases in the sequence shown in Serial No. 1; and

[0079] A gene consisting of a base sequence in which one or more bases are missing, substituted, or added in the base sequence shown in sequence number 2.

[0080] In the aforementioned gene (3), the number of base deletions, substitutions or additions 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, even more preferably 1 to 5.

[0081] The gene mentioned in (3) is preferably a gene consisting of a base sequence in which one or more bases are missing, substituted or added in the base sequence shown in sequence number 1 or 2, and has the function of enhancing or conferring oxygen resistance when the same gene is introduced into Bifidobacterium bacteria.

[0082] (4) A gene consisting of the following DNA base sequence, which is capable of hybridization under stringent conditions with a DNA consisting of a complementary sequence to the base sequence shown in sequence number 1 or 2.

[0083] The Bifidobacterium bacteria of the present invention can have a morphology having any one of the following genes, or a morphology having both of the following genes:

[0084] A gene consisting of a DNA base sequence capable of hybridizing under stringent conditions with a DNA sequence complementary to the base sequence shown in Serial Number 1; and

[0085] A gene is a DNA sequence composed of bases that can hybridize under strict conditions with DNA consisting of a complementary sequence of bases shown in sequence number 2.

[0086] In this specification, "strict conditions" refers to hybridization under moderately or highly strict conditions. Specifically, moderately strict conditions can be readily determined by a person skilled in the art based on the length of the DNA. Basic conditions are illustrated in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Chapters 6-7, Cold Spring Harbor Laboratory Press, 2001. Preferably, under moderately strict conditions, hybridization conditions include 1×SSC to 6×SSC at 42°C to 55°C, more preferably 1×SSC to 3×SSC at 45°C to 50°C, and most preferably 2×SSC at 50°C. When the hybridization solution contains, for example, about 50% formamide, a temperature 5°C to 15°C lower than the above temperatures is used. Washing conditions include 0.5×SSC to 6×SSC at 40°C to 60°C. During hybridization and washing, 0.05% to 0.2%, preferably about 0.1%, of SDS can typically be added. Highly stringent conditions can also be determined readily by those skilled in the art, for example, based on the length of the DNA. Typically, highly stringent conditions include hybridization and / or washing at temperatures higher than moderately stringent conditions and / or at low salt concentrations. For example, hybridization conditions include 0.1×SSC to 2×SSC and 55°C to 65°C, more preferably 0.1×SSC to 1×SSC and 60°C to 65°C, and most preferably 0.2×SSC and 63°C. Washing conditions include 0.2×SSC to 2×SSC and 50°C to 68°C, more preferably 0.2×SSC and 60°C to 65°C.

[0087] The aforementioned gene (4) is preferably a gene composed of a DNA base sequence that can hybridize with DNA composed of a complementary sequence of the base sequence shown in sequence number 1 or 2 under strict conditions, and has the function of enhancing or conferring oxygen resistance when the same gene is introduced into Bifidobacterium bacteria.

[0088] (5) Genes composed of degenerate isomers of the base sequence shown in sequence number 1 or 2.

[0089] The Bifidobacterium bacteria of the present invention can have a morphology having any one of the following genes, or a morphology having both of the following genes:

[0090] A gene composed of degenerate isomers of the base sequence shown in sequence number 1; and

[0091] A gene composed of degenerate isomers of the base sequence shown in sequence number 2.

[0092] (6) Genes encoding proteins consisting of the amino acid sequence indicated by sequence number 3 or 4.

[0093] The protein composed of the amino acid sequence shown in sequence number 3 is SDR (NADH oxidoreductase).

[0094] The protein composed of the amino acid sequence shown in sequence number 4 is a transcriptional regulator.

[0095] The Bifidobacterium bacteria of the present invention can have a morphology having any one of the following genes, or a morphology having both of the following genes:

[0096] The gene encoding a protein consisting of the amino acid sequence shown in sequence number 3; and

[0097] The gene that encodes a protein consisting of the amino acid sequence shown in sequence number 4.

[0098] (7) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in sequence number 3 or 4.

[0099] The Bifidobacterium bacteria of the present invention can have a morphology having any one of the following genes, or a morphology having both of the following genes:

[0100] Genes encoding proteins consisting of amino acid sequences with deletions, substitutions, or additions of one to several amino acids in the amino acid sequence shown in Serial Number 3; and

[0101] Genes that encode proteins consisting of amino acid sequences in which one or more amino acids are missing, substituted, or added in the amino acid sequence shown in sequence number 4.

[0102] In the aforementioned gene (7), the number of amino acid deletions, substitutions, or additions 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.

[0103] The aforementioned gene (7) is preferably a gene that encodes a protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted, or added in the amino acid sequence shown in sequence number 3 or 4, and has the function of enhancing or conferring oxygen resistance when the same gene is introduced into Bifidobacterium bacteria.

[0104] [Cultivation under aerobic conditions]

[0105] The culture method of the present invention includes a step of culturing the Bifidobacterium bacteria of the present invention under aerobic conditions.

[0106] "Aerobic conditions" refer to the conditions under which aerobic bacteria can carry out aerobic respiration. That is, it includes not only aerobic environments where oxygen is present in the culture environment, but also anaerobic environments where there is no oxygen in the culture environment but a certain concentration of dissolved oxygen is present in the culture medium.

[0107] In the cultivation method of the present invention, as an aerobic condition, for example, the oxygen concentration of the cultivation environment is preferably 1% or more, more preferably 3% or more, and even more preferably 6% or more.

[0108] The oxygen concentration in the culture environment can be, for example, less than 21%, preferably less than 18%, and more preferably less than 12%.

[0109] In the cultivation method of the present invention, by adjusting the oxygen concentration in the cultivation environment as described above, the conventional cultivation method, which involves the introduction of anaerobic gases such as carbon dioxide, is no longer required. The cultivation method of the present invention makes the handling of Bifidobacterium bacteria easier than before.

[0110] The Bifidobacterium bacteria cultured using the method of the present invention are easy to handle in an aerobic environment, and therefore can be provided with a high viability rate.

[0111] Furthermore, when the culture method of the present invention is carried out in an anaerobic environment, the dissolved oxygen in the culture medium can also be at a certain concentration or higher.

[0112] Here, "culture environment" refers to the atmosphere surrounding the culture medium in which the Bifidobacterium bacteria of the present invention are cultured.

[0113] After adjusting the oxygen concentration in the culture environment as described above, when culturing the Bifidobacterium bacteria of the present invention in a liquid culture medium, the culture can also be carried out while stirring the aforementioned liquid culture medium.

[0114] By stirring, the efficiency of oxygen uptake from the culture environment into the liquid culture medium increases. Even if the oxygen concentration in the liquid culture medium increases, the Bifidobacterium spp. of the present invention can be cultured without problems due to their oxygen tolerance. Furthermore, by culturing while stirring, nutrients in the liquid culture medium can be supplied evenly, thereby improving the proliferation efficiency of the Bifidobacterium spp. of the present invention.

[0115] After adjusting the oxygen concentration in the culture environment as described above, the Bifidobacterium bacteria of the present invention can also be inoculated on a solid culture medium and cultured.

[0116] In the cultivation method of the present invention, by adjusting the oxygen concentration in the cultivation environment as described above, the conventional cultivation method, which involves the introduction of anaerobic gases such as carbon dioxide, is no longer required. In the cultivation method of the present invention, the handling of Bifidobacterium bacteria becomes easier than existing solid-state cultivation methods.

[0117] There are no particular limitations on the culture medium used to cultivate the *Bifidobacterium* bacteria of this invention; any culture medium commonly used for culturing *Bifidobacterium* bacteria can be used. Specifically, as a carbon source, sugars such as glucose, galactose, lactose, arabinose, mannose, sucrose, fructose, cellobiose, trehalose, starch, starch hydrolysate, and molasses can be used, depending on their assimilability. As a nitrogen source, ammonium salts and nitrates such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate can be used, for example. Furthermore, as inorganic salts, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, potassium sulfate, and ferrous sulfate can be used, for example. Additionally, organic components such as peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract can also be used.

[0118] The dissolved oxygen concentration in the culture medium can be set to 0.01–7 ppm, 0.01–3 ppm, or 0.01–1 ppm.

[0119] In the cultivation method of the present invention, by adjusting the dissolved oxygen concentration in the culture medium as described above, the conventional cultivation method, which involves the introduction of anaerobic gases such as carbon dioxide, is no longer required. The cultivation method of the present invention makes the handling of Bifidobacterium bacteria easier than before.

[0120] Bifidobacteria cultured using the method of this invention are easy to handle in an aerobic environment, and therefore can be provided with a high viability rate.

[0121] The cultivation temperature can be 25–50℃, preferably 35–42℃.

[0122] <Modified Bifidobacteria>

[0123] The present invention also relates to modified Bifidobacterium bacteria that have been enhanced or conferred with oxygen tolerance, having incorporated any one of the genes selected from (1) to (7) above. For the genes in (1) to (7), the description in the section on <culture methods> above applies.

[0124] In this specification, "enhanced" oxygen tolerance refers to a situation where oxygen tolerance is enhanced after the introduction of the gene of this invention, compared to a situation before the introduction of the gene of this invention. Furthermore, in this specification, "conferred" oxygen tolerance refers to a situation where an individual did not possess oxygen tolerance before the introduction of the gene of this invention, but acquired oxygen tolerance through the introduction of the gene of this invention. It should be noted that the strength of oxygen tolerance can be quantitatively evaluated using metrics such as viability and CFU during aerobic culture.

[0125] As a method for introducing the gene of the present invention into Bifidobacteria, known genetic engineering methods can be used without limitation. Competent methods utilizing DNA uptake capacity, electroporation methods utilizing high-voltage pulses, etc., can be used. Furthermore, when embedding the gene of the present invention into the chromosome of Bifidobacteria, methods utilizing homologous recombination or site-specific embedding can be used.

[0126] The modified Bifidobacterium bacteria of the present invention can be cultured even under anaerobic conditions. Furthermore, due to the introduction of the gene of the present invention, they can be cultured even under aerobic conditions.

[0127] The modified Bifidobacterium bacteria of the present invention, in addition to the functions originally possessed by Bifidobacterium bacteria, also have enhanced or endowed with oxygen resistance, and thus can be provided as Bifidobacterium bacteria that are resistant to oxygen stress and have excellent functionality.

[0128] Because the modified Bifidobacterium bacteria of the present invention are easy to manipulate in an aerobic environment, they can be provided with a high viability rate.

[0129] <Activated Bifidobacteria>

[0130] Other aspects of the present invention also involve activated Bifidobacterium bacteria selected from any of the genes upregulated in (1) to (7) above. For the genes in (1) to (7), the description in the section on <culture methods> above applies.

[0131] Here, activated Bifidobacterium refers to a Bifidobacterium bacterium that possesses any one of the genes selected from (1) to (7) above, and that gene is in an upregulated state. In other words, activated Bifidobacterium bacterium is a Bifidobacterium bacterium that possesses any one of the genes selected from (1) to (7) above, and that the expression level of any one of the genes selected from (1) to (7) above is increased.

[0132] When a Bifidobacterium bacterium possessing any one of the genes selected from (1) to (7) above is cultured under aerobic conditions, the gene may be upregulated. That is, whether a Bifidobacterium bacterium is activated can be confirmed by determining the expression level of the gene product (e.g., mRNA, protein) of any one of the genes selected from (1) to (7) above during culture under anaerobic and aerobic conditions. Specifically, by comparing the expression levels of the aforementioned genes when a Bifidobacterium bacterium possessing any one of the genes selected from (1) to (7) above is cultured under anaerobic conditions with those when cultured under aerobic conditions, if the expression of the aforementioned genes increases during aerobic culture, it can be confirmed that the Bifidobacterium bacterium is the activated Bifidobacterium bacterium of the present invention.

[0133] The activated Bifidobacterium bacteria of the present invention can be provided as Bifidobacterium bacteria that are resistant to oxygen stress and have excellent functionality due to the upregulation of genes related to oxygen tolerance.

[0134] The activated Bifidobacterium bacteria of the present invention are easy to handle in an aerobic environment, and therefore can be provided with a high viability rate.

[0135] <Manufacturing Method>

[0136] This invention relates to a method for manufacturing a composition comprising Bifidobacterium, including the above-described culture method. Specifically, the manufacturing method of this invention is characterized by including a step of culturing the Bifidobacterium of this invention under aerobic conditions. In addition to the aforementioned culture step, the manufacturing method of this invention may also include any steps necessary for manufacturing the target composition.

[0137] The manufacturing method of this invention uses Bifidobacterium bacteria with excellent oxygen tolerance. Therefore, not only can the cultivation process be carried out under aerobic conditions, but the entire manufacturing process can also be carried out under aerobic conditions. Consequently, the manufacturing method of this invention does not require the introduction of anaerobic gases such as carbon dioxide throughout the entire manufacturing process, making it easy to operate.

[0138] The composition obtained by the manufacturing method of the present invention is easy to operate in an aerobic environment, and therefore can provide Bifidobacterium bacteria with a high viability rate.

[0139] In the manufacturing method of the present invention, the Bifidobacterium bacteria cultured under aerobic conditions can be the Bifidobacterium bacteria of the present invention, the modified Bifidobacterium bacteria described above, or the activated Bifidobacterium bacteria. Specific methods are as described in the sections on "Modified Bifidobacterium bacteria" and "Activated Bifidobacterium bacteria".

[0140] The “composition” manufactured by the manufacturing method of the present invention includes not only the form of a final product provided to consumers, such as a food composition or a pharmaceutical composition, but also any of the following forms: the Bifidobacterium spp. culture of the present invention after the aforementioned culture process; the form of the additive used to obtain the aforementioned final product; and the form of the intermediate composition generated in the process of obtaining the aforementioned final product.

[0141] The Bifidobacterium spp. bacteria of the present invention contained in the composition manufactured by the manufacturing method of the present invention can be either live or inactivated. As a final product, food compositions or pharmaceutical compositions are preferably exemplified. Food compositions or pharmaceutical compositions can be manufactured by adding live or inactivated Bifidobacterium spp. bacteria of the present invention cultured using the above-described culture method to the raw materials of ordinary food or pharmaceuticals. Except for adding the aforementioned bacteria, they can be manufactured in the same manner as ordinary food or pharmaceuticals. The addition of the aforementioned bacteria can be performed at any stage of the manufacturing process. When live Bifidobacterium spp. bacteria of the present invention are used in the manufacturing process, a final sterilization treatment such as heating or pulverization can be performed to inactivate the Bifidobacterium spp. bacteria of the present invention contained in the composition.

[0142] The composition manufactured by the method of the present invention contains Bifidobacterium bacteria, and therefore, when administered to animals, it is expected to improve the intestinal flora. This composition can be used for therapeutic or non-therapeutic purposes.

[0143] Here, "non-therapeutic purpose" excludes medical procedures, that is, procedures performed on the human body for therapeutic purposes. Examples include health enhancement and cosmetic procedures.

[0144] "Improvement" refers to the improvement of a disease, symptom, or condition; the prevention or delay of the worsening of a disease, symptom, or condition; and the reversal, prevention, or delay of the progression of a disease or symptom.

[0145] "Prevention" refers to preventing or delaying the onset of a disease or symptom in the target population, or reducing the risk of a disease or symptom in the target population.

[0146] When the composition manufactured by the method of the present invention is used for non-therapeutic purposes, it can be administered to healthy individuals. Healthy individuals are defined as those with a healthy gut microbiota who do not suffer from diseases caused by gut microbiota dysbiosis. Examples of diseases caused by gut microbiota dysbiosis include diarrhea and constipation. When the composition manufactured by the method of the present invention is used for non-therapeutic purposes, it can be used to further optimize the healthy gut microbiota possessed by healthy individuals. Furthermore, it can prevent diarrhea or constipation in healthy individuals.

[0147] When the composition manufactured by the method of the present invention is used for therapeutic purposes, it can be administered to unhealthy individuals. Unhealthy individuals include, for example, those suffering from diseases related to gut microbiota. Examples of such diseases include intestinal diseases such as small intestinal tumors, gastrointestinal bleeding, intussusception, gastrointestinal stromal tumors, malabsorption syndrome, Behcet's disease, colorectal cancer, polyps, diverticulitis, acute appendicitis, irritable bowel syndrome, ulcerative colitis, ischemic bowel disease, and Crohn's disease. When the composition manufactured by the method of the present invention is used for therapeutic purposes, it can regulate the gut microbiota of 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 gut microbiota of unhealthy individuals.

[0148] The composition manufactured by the method of the present invention can be used on humans or non-human animals (preferably mammals), more preferably humans and pets, and more preferably humans. Furthermore, the application of this technology is not particularly limited to anyone who desires the effects of probiotics, including, for example, infants, children, adults, middle-aged and elderly people, the elderly, healthy individuals, and those with poor intestinal environments. The present invention is preferably used for infants, adults, the elderly, and those with poor intestinal environments.

[0149] The composition manufactured by the method of the present invention is preferably administered or ingested for at least one week, more preferably for at least four weeks. During this period of administration or ingestion, it is preferably ingested daily.

[0150] There is no particular limitation on the amount of the composition manufactured by the manufacturing method of the present invention. The dry weight of the bacterial cells 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.

[0151] Examples of food compositions include beverages such as formula milk, soft drinks, carbonated drinks, nutritional drinks, fruit juice drinks, and lactic acid bacteria drinks (including concentrated stock solutions and powders for preparation); frozen desserts such as ice cream, fruit syrup ice cream, and shaved ice; confectionery such as sugar, chewing gum, candy, chocolate, compressed sugar, snacks, biscuits, jelly, jam, cream, and baked goods; dairy products such as processed milk, milk beverages, fermented milk, drinkable yogurt, and butter; bread; enteral nutrition foods, liquid diets such as porridge, weaning foods, and sports drinks; and other functional foods. Additionally, the food composition can be a supplement, such as a tablet supplement. When used as a supplement, the Bifidobacterium bacteria of this invention can be ingested without affecting other dietary intake and calorie consumption for each day's food intake. Examples of commercially available foods other than those mentioned above include baby food, rice toppings, and ochazuke (rice with kelp).

[0152] It should be noted that the aforementioned formula milk includes formula milk powder and formula liquid milk. Formula milk powder is defined in the Ministry Ordinance on the composition standards of milk and dairy products (Milk, etc. Ordinance) as "a food made by processing fresh milk, cow's milk, special milk or food made from them, or by using them as the main raw materials and adding nutrients required by infants and young children into a powder form".

[0153] For formula liquid milk, the aforementioned regulation defines it as "a liquid food made by processing fresh milk, cow's milk, special milk, or food made from them, or by using them as the main raw material and adding nutrients required by infants and young children." Formula milk refers to milk that is mixed with various proteins, fats, carbohydrates, minerals, vitamins, and other nutrients, and also includes formula milk that has been processed into powder or liquid form.

[0154] In addition, formula milk powder also includes: "infant formula milk powder", "infant formula liquid milk", and "milk powder for pregnant and lactating women" as special purpose foods as stipulated in the Health Promotion Law; infant formula milk powder for infants aged 0-12 months; formula milk powder for older infants aged 6-9 months and younger children (under 3 years old); formula milk powder for newborns with a birth weight of less than 2500g (low birth weight infants); various therapeutic milk powders for infants and young children suffering from milk allergy, lactose intolerance, and other diseases; formula milk powder for toddlers; nutritional powder for adults; and nutritional powder for the elderly.

[0155] Food compositions may be used for any purpose, whether therapeutic or non-therapeutic, but are preferably used for non-therapeutic purposes on healthy individuals.

[0156] The pharmaceutical composition may be for oral administration. The dosage form of the pharmaceutical composition is not particularly limited. For oral administration, it can be formulated as solid dosage forms such as powders, granules, tablets, tablet lozenges, and capsules; or as liquid dosage forms such as solutions, syrups, suspensions, and emulsions. For non-oral administration, it can be formulated as suppositories, sprays, inhalers, ointments, patches, and injections. Furthermore, during formulation, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring and odor-correcting agents, diluents, surfactants, or solvents for injections, which are commonly used as formulation carriers, can be used. These components can be appropriately selected by those skilled in the art depending on the dosage form.

[0157] The pharmaceutical composition may be used for any purpose, whether therapeutic or not, but is preferably used for therapeutic purposes on non-healthy individuals.

[0158] When manufacturing food or pharmaceutical compositions using the manufacturing method of the present invention, the content of Bifidobacterium cells of the present invention in the composition, based on the daily intake, is preferably 1 × 10⁻⁶. 5 ~1×10 12 CFU / kg body weight / day, preferably 1×10 7 ~1×10 11 CFU / kg body weight / day, further optimized to 1×10 8 ~1×10 10 CFU / kg body weight / day. Alternatively, the dosage per individual (body weight) (in other words, the amount given), preferably 10. 7 ~10 14 CFU / day, preferably 10 8 ~10 13 CFU / day, further optimized to 10 9 ~10 12 CFU / day. When the Bifidobacterium bacteria are inactivated, CFU can be replaced with individual cells.

[0159] The composition manufactured using the method of the present invention preferably contains a prebiotic. Examples of prebiotics include indigestible dextrin, inulin, lactulose, galactooligosaccharides, fructooligosaccharides, soybean oligosaccharides, lactulose oligosaccharides, xylooligosaccharides, isomaltooligosaccharides, raffinose, coffee-derived mannose, gluconic acid, polydextrose, guar gum metabolites, alginate, pectin, isomaltooligosaccharides, barley β-glucan, and human milk oligosaccharides (HMOs). Examples of human milk oligosaccharides include 2'-fucosyllactose, 3'-sialyllactose, lactoyl-N-tetrasaccharide 3-difucosyllactose, 3-fucosyllactose, 3-fucosyl-3'-sialyllactose, lactoyl-N-fucopentose I, lactoyl-N-fucopentose II, lactoyl-N-fucopentose III, lactoyl-N-fucopentose V, lactoyl-N-difucosylhexose I, lactoyl-N-difucosylhexose II, lactoyl-N-sialylpentose, LSTa, LSTb, and LSTc.

[0160] In one embodiment of the manufacturing method of the present invention, the following step is included: drying a bacterial solution containing the *Bifidobacterium* bacteria of the present invention obtained by culturing based on the aforementioned culturing method. According to the above embodiment, a *Bifidobacterium* bacterial powder of the present invention can be obtained.

[0161] In the manufacturing method of the present invention, since Bifidobacterium bacteria with excellent oxygen tolerance are used, the drying process can be carried out under aerobic conditions. The manufacturing method of the present invention provides a composition with a high viability rate of Bifidobacterium bacteria even after the drying process.

[0162] There are no particular restrictions on drying methods; examples include freeze drying and spray drying.

[0163] In the bacterial powder, the cells of the Bifidobacterium spp. of the present invention can be broken down. This broken material can be obtained by breaking down live bacteria, by breaking down inactivated bacteria, or by subjecting the broken down material to heating, freeze-drying, or other methods. From the viewpoint of increasing the viability rate, freeze-drying the culture of the Bifidobacterium spp. of the present invention is suitable; conversely, from the viewpoint of improving production efficiency, spray drying the aforementioned culture is suitable.

[0164] In one embodiment, a food composition is manufactured by adding the bacterial powder obtained from the above-described drying process to the raw material composition. The food composition that can be manufactured in this embodiment is as described above. The raw material composition can be appropriately selected based on the food composition being manufactured. An embodiment may involve adding the bacterial powder itself to the raw material composition, or an embodiment may involve adding a solution obtained by dissolving the bacterial powder in water or the like to the raw material composition.

[0165] In the manufacturing method of the present invention, the food composition is manufactured by adding the bacterial powder obtained in the above-mentioned drying process to the raw material composition, thereby providing a food composition with a high viability rate of Bifidobacterium bacteria.

[0166] Furthermore, food compositions can also be manufactured through a fermentation process based on the added Bifidobacterium bacteria of the present invention. Examples of such food compositions include lactic acid bacteria beverages and fermented milk.

[0167] In one embodiment, the method further includes adding a starter culture comprising Bifidobacterium bacteria obtained by the aforementioned culture method to the raw material composition, and then fermenting. This embodiment is effective, for example, in the manufacture of lactic acid bacteria beverages or fermented milk.

[0168] In addition to the *Bifidobacterium* bacteria of the present invention, the aforementioned starter culture may also contain other bacterial species. Among these other species, lactic acid bacteria are preferred; specifically, examples include *Lactococcus lactis*, *Streptococcus thermophilus*, and *Lactobacillus delbrueckii*. Subspecies are not particularly limited; examples of *Lactococcus lactis* include *Lactococcus lactis* subsp. *lactolaccos* and *Lactococcus lactis* subsp. *fat*. Additionally, examples of *Lactobacillus delbrueckii* include *Lactobacillus delbrueckii* subsp. *bulgaricus*.

[0169] The composition manufactured by the method of the present invention can also be provided as an article contained in a packaging container providing an aerobic environment. Since the Bifidobacterium bacteria of the present invention are easy to handle in an aerobic environment, a high viability rate can be maintained even when they are provided in a packaging container providing an aerobic environment.

[0170] <Filtering Method>

[0171] The present invention also relates to a method for screening Bifidobacterium bacteria with oxygen tolerance, the method comprising the step of selecting Bifidobacterium bacteria using any one of the genes selected from (1) to (7) above as an indicator.

[0172] As described above, Bifidobacteria possessing the gene of the present invention exhibit oxygen tolerance, enabling them to be cultured under aerobic conditions. In other words, Bifidobacteria possessing the gene of the present invention can be determined to be oxygen-tolerant.

[0173] The screening method of the present invention can be implemented as follows: If the *Bifidobacterium* bacteria to be tested possesses any one of the following genes, it is determined to be oxygen-resistant; or, if it possesses both of the following genes, it is determined to be oxygen-resistant.

[0174] A gene consisting of the base sequence shown in sequence number 1; and

[0175] A gene consisting of the base sequence shown in sequence number 2.

[0176] The screening method of the present invention can be implemented as follows: If the *Bifidobacterium* bacteria to be tested possesses any one of the following genes, it is determined to be oxygen-resistant; or, if it possesses both of the following genes, it is determined to be oxygen-resistant.

[0177] Genes that share more than 90% identity with the base sequence shown in sequence number 1; and

[0178] Genes that share more than 90% identity with the base sequence shown in sequence number 2.

[0179] The screening method of the present invention can be implemented as follows: If the *Bifidobacterium* bacteria to be tested possesses any one of the following genes, it is determined to be oxygen-resistant; or, if it possesses both of the following genes, it is determined to be oxygen-resistant.

[0180] A gene consisting of a base sequence in which one or more bases are deleted, substituted, or added in the base sequence shown in sequence number 1;

[0181] A gene consisting of a base sequence in which one or more bases are missing, substituted, or added in the base sequence shown in sequence number 2.

[0182] The screening method of the present invention can be implemented as follows: If the *Bifidobacterium* bacteria to be tested possesses any one of the following genes, it is determined to be oxygen-resistant; or, if it possesses both of the following genes, it is determined to be oxygen-resistant.

[0183] A gene consisting of the following DNA base sequence, said DNA capable of hybridizing under stringent conditions with DNA consisting of a complementary sequence to the base sequence shown in Serial Number 1; and

[0184] A gene consisting of the following DNA base sequence, which can hybridize under stringent conditions with DNA consisting of a complementary sequence of the base sequence shown in sequence number 2.

[0185] The screening method of the present invention can be implemented as follows: If the *Bifidobacterium* bacteria to be tested possesses any one of the following genes, it is determined to be oxygen-resistant; or, if it possesses both of the following genes, it is determined to be oxygen-resistant.

[0186] A gene composed of degenerate isomers of the base sequence shown in sequence number 1; and

[0187] A gene composed of degenerate isomers of the base sequence shown in sequence number 2.

[0188] The screening method of the present invention can be implemented as follows: If the *Bifidobacterium* bacteria to be tested possesses any one of the following genes, it is determined to be oxygen-resistant; or, if it possesses both of the following genes, it is determined to be oxygen-resistant.

[0189] The gene encoding a protein consisting of the amino acid sequence shown in sequence number 3; and

[0190] The gene that encodes a protein consisting of the amino acid sequence shown in sequence number 4.

[0191] The screening method of the present invention can be implemented as follows: If the *Bifidobacterium* bacteria to be tested possesses any one of the following genes, it is determined to be oxygen-resistant; or, if it possesses both of the following genes, it is determined to be oxygen-resistant.

[0192] A gene encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in sequence number 3; and

[0193] A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted, or added in the amino acid sequence shown in sequence number 4.

[0194] For the genes mentioned in (1) to (7) that serve as indicators of the screening method of the present invention, the descriptions set forth in the aforementioned <culture method> section shall apply.

[0195] To implement the screening method of the present invention, there are no particular limitations on the method used to determine the presence or absence of the gene of the present invention. The presence or absence of the gene of the present invention can be determined by using genomic DNA as the target, or indirectly by measuring the presence or absence of mRNA as its transcription product or protein as its translation product.

[0196] For example, primers capable of specific hybridization with the base sequence region of the gene of the present invention can be designed, and the determination can be made by PCR using the genome of the Bifidobacterium genus of the test subject as a template.

[0197] Alternatively, mRNA can be extracted from Bifidobacterium bacteria in the test subject, and RT-PCR or arrays that can specifically detect the transcript of the gene of the present invention can be used to indirectly determine the presence or absence of the gene of the present invention.

[0198] Alternatively, antibodies targeting the protein encoded by the gene of the present invention can be used to indirectly determine the presence or absence of the gene of the present invention through methods such as Western blotting.

[0199] In the screening method of the present invention, the genes (1) to (7) above are used as indicators to easily screen out Bifidobacterium bacteria with excellent oxygen tolerance.

[0200] <Composition for Starter Culture>

[0201] The present invention also relates to a composition for starter cultures comprising Bifidobacterium bacteria having any one of the genes selected from (1) to (7) above. For the genes (1) to (7), the description in the section on <culture method> above applies.

[0202] The starter culture composition of the present invention can be obtained by inoculating and culturing the Bifidobacterium genus of the present invention in a culture medium.

[0203] In the invention of the composition for starter cultures, modified Bifidobacterium bacteria can be used as the Bifidobacterium bacteria of the present invention. That is, in another embodiment of the invention of the composition for starter cultures, modified Bifidobacterium bacteria with enhanced or conferred oxygen tolerance are included, which have been introduced with any one of the genes selected from (1) to (7) above. For modified Bifidobacterium bacteria, the description in the item <Modified Bifidobacterium bacteria> above applies.

[0204] In the description of the composition for starter cultures, activated Bifidobacterium bacteria can be used as the Bifidobacterium bacteria of the present invention. That is, in another embodiment of the invention of the composition for starter cultures, activated Bifidobacterium bacteria selected from any one of the gene-upregulated activated Bifidobacterium bacteria described in (1) to (7) above are included. For activated Bifidobacterium bacteria, the description in the aforementioned item <Activated Bifidobacterium bacteria> applies.

[0205] The starter culture composition of the present invention can be in the form of a fermented milk starter composition. The fermented milk starter composition can be obtained by inoculating and culturing the Bifidobacterium spp. of the present invention in a milk culture medium composed of whole milk, skim milk or reconstituted milk, or a synthetic or semi-synthetic culture medium with lactose or glucose as the main components.

[0206] If the starter culture composition of the present invention is used to manufacture fermented milk such as drinkable yogurt and acidic milk beverages, a fermented composition with a high viable count of Bifidobacterium bacteria of the present invention can be obtained.

[0207] The starter culture composition of the present invention may include lactic acid bacteria in addition to the Bifidobacterium spp. of the present invention. Specific details of this embodiment will be described in detail in the "Composition" section below.

[0208] <Composition>

[0209] The present invention also relates to a composition comprising:

[0210] Bifidobacteria possessing any one of the genes selected from (1) to (7) above; and

[0211] It is selected from one or more lactic acid bacteria from Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus delbrueckii.

[0212] For genes (1) to (7), the description in the aforementioned <Cultivation Methods> section applies.

[0213] In the invention of the composition, modified Bifidobacterium bacteria can be used as the Bifidobacterium bacteria of the present invention.

[0214] That is, in other embodiments of the invention, the composition comprises: a modified Bifidobacterium bacterium introduced with any one of the genes selected from (1) to (7) above, which enhances or confers oxygen tolerance; and

[0215] It is selected from one or more lactic acid bacteria from Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus and Lactobacillus delbrueckii.

[0216] For modified Bifidobacterium bacteria, the instructions in the aforementioned project "Modified Bifidobacterium bacteria" shall apply.

[0217] In the invention of the composition, activated Bifidobacterium bacteria can be used as the Bifidobacterium bacteria of the present invention.

[0218] That is, in other embodiments of the invention, the composition comprises:

[0219] Selected from any one of the gene-upregulated activated Bifidobacterium bacteria from (1) to (7) above; and

[0220] It is selected from one or more lactic acid bacteria from Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus delbrueckii.

[0221] For activated Bifidobacteria, the description in the aforementioned section on "Activated Bifidobacteria" applies.

[0222] The Bifidobacterium bacteria and the aforementioned lactic acid bacteria of the present invention possess aerobic tolerance, allowing them to be cultured even under aerobic conditions. That is, the composition of the present invention is a mixture of Bifidobacterium and lactic acid bacteria that can be cultured even under aerobic conditions. Due to this property, the composition of the present invention is useful as a starter culture.

[0223] If the composition of the present invention is used as a starter culture, it is possible to provide a fermentation composition of the present invention with a high viability of Bifidobacterium and lactic acid bacteria.

[0224] The composition of the present invention can be in the form of a fermented milk starter composition. The fermented milk starter composition can be obtained by inoculating and culturing the Bifidobacterium spp. of the present invention in a milk culture medium composed of whole milk, skim milk or reconstituted milk, or a synthetic or semi-synthetic culture medium with lactose or glucose as the main components.

[0225] When the composition of the present invention is used as a starter for fermenting milk to produce fermented milk such as drinkable yogurt and acidic milk beverages, a fermented composition with a high viable count of Bifidobacterium and lactic acid bacteria can be obtained.

[0226] Example

[0227] <Experimental Example>

[0228] (1) The Bifidobacterium bacteria used

[0229] In this experimental case, the following eight strains of Bifidobacterium bacteria were used.

[0230] MCC02042 (Example 1): Bifidobacterium longum infantis subsp. as identified by NITE BP-03068;

[0231] MCC10110 (Example 2): Bifidobacterium longum subsp. infantis. A strain held by the Research and Information Center of Morinaga Milk Industry Co., Ltd. (5-1-83 Higashihara, Zama City, Kanagawa Prefecture 252-8583, Japan).

[0232] JCM1210: Bifidobacterium longum infantile subspecies identified by JCM number JCM1210;

[0233] JCM1222: Bifidobacterium longum infantile subspecies identified by JCM number JCM1222;

[0234] JCM1260: Bifidobacterium longum infantile subspecies identified by JCM number JCM1260;

[0235] JCM11344: Bifidobacterium longum infantile subspecies identified by JCM number JCM11344;

[0236] JCM11345: Bifidobacterium longum infantile subspecies identified by JCM number JCM11345;

[0237] JCM11347: Bifidobacterium longum infantile subspecies identified by JCM number JCM11347;

[0238] (2) Oxygen resistance test

[0239] Each bacterial strain (MCC2042, MCC10110, JCM1210, JCM1222, JCM1260, JCM11344, JCM11345, JCM11347) was cultured anaerobically at 37°C for 16 hours on MRS medium (BD Difco). 100 μL of the cultured bacterial suspension was then spread onto BL agar medium (Eiken Chemical Co., Ltd.) and incubated aerobically at 37°C for 48 hours. Colony formation on BL agar was evaluated. The results showed that only MCC2042 and MCC10110 formed colonies; the other strains did not. Based on these results, MCC2042 and MCC10110 were determined to be oxygen-tolerant strains.

[0240] (3) Draft genome sequencing

[0241] Each bacterial strain (JCM1210, JCM1222, JCM1260, JCM11344, JCM11345, JCM11347) was cultured in MRS medium at 37°C under anaerobic conditions for 16 hours. The culture medium was centrifuged to obtain bacterial pellets. DNA extraction was performed using the DNeasy Blood and Tissue Kit (QIAGEN). Genomic library preparation was performed using 1 ng of extracted DNA using the Nextera XT DNA Sample Preparation Kit (Illumina). After PCR-based amplification and purification (Thermofisher Scientific, Veriti 200), the fragment size distribution of the tagged DNA was analyzed using an Agilent 2100 Bioanalyzer and a high-sensitivity DNA analysis kit (Agilent).

[0242] For the library, sequencing analysis was performed using the MiSeq Personalized Sequencing System and MiSeq Sequencing Kit v2 (500 cycles). The obtained sequencing data were pruned and assembled using the CLC Genomics Workbench (QIAGEN). The default settings were used, but contiguous groups below 2000 bp were removed.

[0243] (4) Complete genome sequence

[0244] Each bacterial strain (MCC2042, MCC10110) was cultured in MRS medium at 37°C under anaerobic conditions for 16 hours. The culture was centrifuged to obtain bacterial pellets. DNA was extracted using the NucleoBond High Molecular Weight (HMW) DNA Kit (Machery-Nagel). Genomic library preparation was performed using the SMRTbell Rapid Template Preparation Kit (Pacific Biosciences). Size distribution was confirmed by pulsed-field electrophoresis, and size selection was performed using BluePippi (Sage Science). Sequencing analysis was performed using PacBio RSII and SMRT Cell 8 Pac V3 as long-read sequencers. The obtained sequencing data was assembled using Canu. Default settings were used.

[0245] (5) Comparative genomics analysis

[0246] Using the obtained complete genome and draft genome FATA files as input, and annotating them with Bakta, FAA files were obtained. Using all obtained FAA files as input, sonic paranoid2 (Non-Patent Literature 1) was used to obtain an ortholog table. The specified parameters were --mmseqs 7.5 and --go. From the obtained ortholog table, orthologs existing only in MCC2042 and MCC10110 and not in other strains were extracted, obtaining a list of amino acid sequences of genes from MCC2042 included in the orthologs. These amino acid sequences were then predicted using CLEAN (Non-Patent Literature 2) based on their EC numbers (Enzyme Commission numbers).

[0247] As shown in (2) above, MCC2042 and MCC10110 were able to form colonies under aerobic conditions. It is believed that these two strains have a higher ability to decompose oxygen than other strains. That is, it is speculated that the responsible gene for the oxygen tolerance of these two strains is the gene encoding an oxidoreductase (enzymes whose EC number begins with 1. Hereinafter, it will also be referred to as "enzymes whose EC number begins with 1").

[0248] Based on this inference, the inference results of the amino acid sequence list and EC number obtained from the above comparative genomic analysis were confirmed. As a result, genes consisting of the base sequence shown in sequence number 1 or 2 were identified as genes that are not possessed by other strains but are only possessed by MCC2042 and MCC10110 and that encode enzymes (oxidoreductases) with EC numbers starting with 1.

[0249] It should be noted that for genes not possessed by other strains but only by MCC2042 and MCC10110, other than those consisting of the base sequence shown in sequence number 1 or 2, a careful examination was conducted based on the inferred EC number, but no reasonable deduction could be made as to their association with oxygen tolerance.

[0250] In summary, the following conclusions can be drawn: In the above (2) experiments, the responsible genes for oxygen tolerance identified only in MCC2042 and MCC10110 are genes composed of the base sequence shown in sequence number 1 or 2.

[0251] <Manufacturing Example 1>

[0252] Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068) was added to 3 mL of MRS liquid medium and cultured aerobically 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 this composition was dissolved in 200 g of water to obtain a composition containing Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068).

[0253] <Manufacturing Example 2>

[0254] Skim milk powder, cream, and milk protein were mixed and dissolved to prepare a 50L milk raw material consisting of 3.0% (w / w) milk fat and 12.0% (w / w) non-fat milk solids. The mixture was heated to 70°C, homogenized at 15 MPa, sterilized at 90°C for 10 minutes, and cooled to 40°C. This sterilized milk raw material was inoculated with 500mL of a culture of *Lactococcus lactis* subsp. *lactotrichum* and 1.5 × 10⁻⁶ frozen cells of *Bifidobacterium longum* subsp. *infantica* MCC02042 (NITE BP-03068) (manufactured by Morinaga Milk Industry Co., Ltd.). 14 The fermented milk was obtained by incubating CFU (colony forming unit) and 0.002% of yogurt starter containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus at 37°C for 4 hours. 100g of this fermented milk was then filled into a 120mL paper container (manufactured by Singi, 71mm diameter, 56mm height), sealed with an aluminum foil lid, and cooled to 10°C. This yielded a plain-flavored fermented dairy product.

[0255] <Manufacturing Example 3>

[0256] Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068) was added to 3 mL of MRS liquid medium and cultured at 37 °C under aerobic conditions for 24 hours. The culture solution was then concentrated and freeze-dried to obtain freeze-dried bacterial powder (bacterial powder).

[0257] 10 kg of demineralized milk whey protein powder (Mirai Co., Ltd.), 6 kg of milk casein powder (Fonterra Co., Ltd.), 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 galacto-oligosaccharide liquid sugar (Yakult Pharmaceutical Industry Co., Ltd.) were dissolved in 300 kg of warm water and then heated at 90°C for 10 minutes. 28 kg of formulated fat (Taiyo Oil Co., Ltd.) was added and homogenized. Then, sterilization and concentration processes were performed, followed by spray drying to prepare approximately 95 kg of formula milk powder. 100g of the freeze-dried powder of the above-mentioned Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068) which had been dispersed with starch was added to it (1.8×10⁻⁶). 11 Approximately 95 kg of formula milk powder containing bifidobacteria and oligosaccharides was prepared by dissolving the obtained formula milk powder in water to prepare a standard formula milk liquid concentration, i.e., a total solids concentration of 14% (w / v). The bifidobacteria count in the formula milk liquid was 2.7 × 10⁻⁶. 9 cfu / 100mL.

[0258] <Reference Example>

[0259] Temperature-sensitive plasmids developed for Bifidobacterium genetic modification were used, and sequences 1 and 2, which are responsible genes for oxygen tolerance, were deleted based on Non-Patent Literature 3. 500-1000 bases upstream and downstream of each of sequences 1 and 2 were selected and amplified by PCR. Primers with an overlapping sequence of 15-25 bases added to the 5' end were used to facilitate the binding of the temperature-sensitive plasmid to the target sequence and the binding of the upstream and downstream target sequences to each other based on the In-Fusion reaction. The temperature-sensitive plasmid was linearized by restriction enzyme digestion. The linearized plasmid was mixed with the previously amplified base sequence and bound using In-Fusion Snap AssemblyMaster Mix (TaKaRa Bio). The resulting plasmid was transformed into DH5α (TaKaRa Bio) and amplified. The amplified plasmid was extracted and introduced into Bifidobacterium infantis MCC02042 by electroporation. Electroporated Bifidobacterium infantis MCC02042 was cultured on GAM agar containing spectinomycin at 30°C. To facilitate single crossover, plasmid-transformed Bifidobacterium infantis MCC02042 was cultured on Gifu anaerobic agar (GAM) containing spectinomycin at temperatures above 39°C and below 42°C. Colonies were scraped and cultured at 30°C for 1–2 days on a medium containing 0.05% cysteine ​​from the de Man, Rogosa, and Sharpe (MRS). The bacterial culture was then spread onto MRS agar and cultured at 37°C for 1 day. Colonies were then replicated and cultured at 37°C. After culturing, colonies that did not increase in size were selected, and PCR was used to confirm the deletion of sequences 1 and 2, which are genes associated with oxygen tolerance. In the case of a deletion strain, the deletion strain was cultured in BL medium at 37°C for about 72 hours under aerobic conditions, and the growth difference was compared with that of the wild-type strain (MCC2042) to evaluate whether sequence numbers 1 and 2 are responsible genes for oxygen tolerance.

[0260] Industrial availability

[0261] This invention can be applied to the manufacturing technology of food or pharmaceuticals containing oxygen-resistant Bifidobacterium bacteria.

Claims

1. A method for culturing Bifidobacterium bacteria, comprising the step of culturing Bifidobacterium bacteria having any one of the genes selected from (1) to (7) under aerobic conditions: (1) A gene consisting of the base sequence indicated by sequence number 1 or 2; (2) Genes that have more than 90% identity with the base sequence shown in sequence number 1 or 2; (3) A gene consisting of a base sequence in which one or more bases are missing, substituted or added in the base sequence shown in sequence number 1 or 2; (4) A gene consisting of the base sequence of a DNA that can hybridize under strict conditions with a DNA consisting of a complementary sequence of the base sequence shown in sequence number 1 or 2; (5) Genes consisting of degenerate isomers of the base sequence shown in sequence number 1 or 2; (6) Genes that encode proteins consisting of the amino acid sequence indicated by sequence number 3 or 4; (7) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted or added in the amino acid sequence shown in sequence number 3 or 4.

2. The cultivation method according to claim 1, wherein, The Bifidobacterium species mentioned is Bifidobacterium longum.

3. The cultivation method according to claim 1, wherein, The Bifidobacterium species mentioned is Bifidobacterium longum infantis subspecies.

4. The cultivation method according to claim 1, wherein, The Bifidobacterium species mentioned is Bifidobacterium longum subsp. infantis MCC02042 (NITE BP-03068).

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

6. The manufacturing method according to claim 5, wherein, The manufacturing method includes the following steps: drying the bacterial solution containing the Bifidobacterium bacteria obtained by the culture method. The composition is a bacterial powder obtained by the drying process or a food composition with the bacterial powder added.

7. The manufacturing method according to claim 5, wherein, The manufacturing method includes the following steps: adding a starter culture containing Bifidobacterium bacteria to the raw material composition and fermenting it, wherein the Bifidobacterium bacteria are obtained by culturing based on the culture method.

8. A modified Bifidobacterium bacterium that has been enhanced or conferred with oxygen tolerance, having incorporated any one of the genes selected from (1) to (7) below. (1) A gene consisting of the base sequence indicated by sequence number 1 or 2; (2) Genes that have more than 90% identity with the base sequence shown in sequence number 1 or 2; (3) A gene consisting of a base sequence in which one or more bases are missing, substituted or added in the base sequence shown in sequence number 1 or 2; (4) A gene consisting of the base sequence of a DNA that can hybridize under strict conditions with a DNA consisting of a complementary sequence of the base sequence shown in sequence number 1 or 2; (5) Genes consisting of degenerate isomers of the base sequence shown in sequence number 1 or 2; (6) Genes that encode proteins consisting of the amino acid sequence indicated by sequence number 3 or 4; (7) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted or added in the amino acid sequence shown in sequence number 3 or 4.

9. A method for screening aerobic Bifidobacterium bacteria, comprising the step of selecting Bifidobacterium bacteria using any one of the following (1) to (7) as an indicator: (1) A gene consisting of the base sequence indicated by sequence number 1 or 2; (2) Genes that have more than 90% identity with the base sequence shown in sequence number 1 or 2; (3) A gene consisting of a base sequence in which one or more bases are missing, substituted or added in the base sequence shown in sequence number 1 or 2; (4) A gene consisting of the base sequence of a DNA that can hybridize under strict conditions with a DNA consisting of a complementary sequence of the base sequence shown in sequence number 1 or 2; (5) Genes consisting of degenerate isomers of the base sequence shown in sequence number 1 or 2; (6) Genes that encode proteins consisting of the amino acid sequence indicated by sequence number 3 or 4; (7) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted or added in the amino acid sequence shown in sequence number 3 or 4.

10. A composition for starter cultures comprising a Bifidobacterium bacterium having any one of the genes selected from (1) to (7) below, (1) A gene consisting of the base sequence indicated by sequence number 1 or 2; (2) Genes that have more than 90% identity with the base sequence shown in sequence number 1 or 2; (3) A gene consisting of a base sequence in which one or more bases are missing, substituted or added in the base sequence shown in sequence number 1 or 2; (4) A gene consisting of the base sequence of a DNA that can hybridize under strict conditions with a DNA consisting of a complementary sequence of the base sequence shown in sequence number 1 or 2; (5) Genes consisting of degenerate isomers of the base sequence shown in sequence number 1 or 2; (6) Genes that encode proteins consisting of the amino acid sequence indicated by sequence number 3 or 4; (7) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted or added in the amino acid sequence shown in sequence number 3 or 4.

11. A composition comprising: a Bifidobacterium bacterium having a gene selected from (1) to (7) below, and one or more lactic acid bacteria selected from Lactococcus lactis, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus delbrueckii. (1) A gene consisting of the base sequence indicated by sequence number 1 or 2; (2) Genes that have more than 90% identity with the base sequence shown in sequence number 1 or 2; (3) A gene consisting of a base sequence in which one or more bases are missing, substituted or added in the base sequence shown in sequence number 1 or 2; (4) A gene consisting of the base sequence of a DNA that can hybridize under strict conditions with a DNA consisting of a complementary sequence of the base sequence shown in sequence number 1 or 2; (5) Genes consisting of degenerate isomers of the base sequence shown in sequence number 1 or 2; (6) Genes that encode proteins consisting of the amino acid sequence indicated by sequence number 3 or 4; (7) A gene encoding a protein consisting of an amino acid sequence in which one or more amino acids are missing, substituted or added in the amino acid sequence shown in sequence number 3 or 4.

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