Bacteria, compositions and methods for making same, and prebiotic compositions

Abstract

The present invention relates to bacteria, compositions and methods for their production, and prebiotic compositions. By the present invention, novel Bifidobacterium bacteria having assimilation ability of two or more HMOs are provided. The bacteria are selected from the group consisting of Bifidobacterium longum subsp. infantis (NITE BP-03068), Bifidobacterium bifidum (NITE BP-03058), and Bifidobacterium bifidum (NITE BP-03067).

Description

[0001] This application is a divisional application of the invention patent application filed on January 19, 2021, with application number 202180009511.8 and entitled "Bacteria, Compositions and Methods for Manufacturing the Same Thereof, and Prebiotic Compositions". Technical Field

[0002] This invention relates to bacteria, compositions thereof, methods of manufacturing thereof, and prebiotic compositions.

[0003] This application claims priority based on Japanese Patent Application No. 2020-007102 filed in Japan on January 20, 2020, the contents of which are incorporated herein by reference. Background Technology

[0004] In recent years, research on probiotics and prebiotics—components that promote the growth of beneficial bacteria (also known as "beneficiary bacteria")—has flourished, aiming to regulate the intestinal environment by actively consuming bacteria that have a positive effect on animals. Probiotics refer to bacteria that play a beneficial role in the gut, while prebiotics refer to substances that serve as selective nutrient sources for these probiotics and promote their growth. It is known that prebiotics can provide beneficial effects on human health, such as promoting the growth of lactobacilli / Bifidobacterium bacteria, regulating the intestines, and preventing / improving inflammatory bowel disease.

[0005] Human milk oligosaccharides (hereinafter also referred to as "HMOs") comprise 10–20 g / L in breast milk and are said to be a mixture of more than 130 oligosaccharides. Their structure consists of 13 core structures with the addition of fucose and sialic acid. Human milk has a high proportion of fucosylated neutral sugars, representative examples being 2'-fucosyllactose, lactose-N-fucopentose I, lactose-N-difucohexasose I, and lactose-N-tetrasaccharide. These four oligosaccharides alone account for 1 / 3 to 1 / 4 of all human milk oligosaccharides. Oligosaccharides containing lactose-N-disaccharide are called type I, and those containing N-acetyllactosamine (LacNAc) are called type II. The content of type I lactose-N-tetrasaccharide and lactose-N-fucopentose I in human milk is higher than that of type II lactose-N-neotetrasaccharide and lactose-N-fucopentose III. The coexistence of type I and type II, and the dominance of type I, are characteristics of human milk oligosaccharides that distinguish them from those of other species (Non-Patent Literature 1 and 2).

[0006] HMOs contained in breast milk, namely 2'-FL and LNT, have bifidobacterial proliferative activity. It is known that 2'-FL and LNT act as growth factors for bifidobacteria by selectively targeting them, and are important for the formation of a bifidobacterial-dominant gut microbiota. Furthermore, HMOs are in a state of being synthesizable and industrially usable (Patent Document 1).

[0007] Bifidobacteria utilize the bifidobacteria-specific GNB / LNB pathway to metabolize a portion of HMOs. Many species of bifidobacteria commonly found in the human gut possess this pathway, but almost none of the bifidobacteria commonly found in the intestines of animals and insects outside of humans possess it. Representative examples of bifidobacteria capable of using HMOs as their sole carbon source for growth include four species commonly found in infants: *Bifidobacterium longum subspecies longum*, *Bifidobacterium longum subspecies infantis*, *Bifidobacterium breve*, and *Bifidobacterium bifidum*. In particular, the utilization of various HMOs by *Bifidobacterium longum subspecies infantis* and *Bifidobacterium bifidum* has been reported (Non-Patent Literature 3).

[0008] Existing technical documents

[0009] Patent documents

[0010] Patent Document 1: International Publication No. 2014 / 086373

[0011] Non-patent literature

[0012] Non-patent literature 1: Japanese Journal of Lactic Acid Bacteria Vol. 22, No. 1, pp. 15-25, 2011

[0013] Non-patent literature 2: Milk Science Vol. 56, No. 4, pp155-176, 2008

[0014] Non-patent literature 3: Milk Science Vol. 61, No.2, pp115-124, 2012 Summary of the Invention

[0015] The problem the invention aims to solve

[0016] As mentioned earlier, some Bifidobacteria do not have the ability to assimilate HMOs or have a low assimilation ability when metabolizing HMOs. On the other hand, some Bifidobacteria, such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum, also have the ability to assimilate more than two HMOs.

[0017] The objective of this invention is to provide novel Bifidobacterium bacteria with the ability to assimilate two or more HMOs.

[0018] Solution for solving the problem

[0019] First, in this invention, at least one bacterium selected from (a) to (c) below is provided.

[0020] (a) Bifidobacterium longum infantis subspecies (NITE BP-03068)

[0021] (b) Bifidobacterium bifidum (NITE BP-03058)

[0022] (c) Bifidobacterium bifidum (NITE BP-03067)

[0023] The bacteria selected from (a) to (c) above have the ability to assimilate 2'-fucosylated lactose, 3'-sialyl lactose, and lactose-N-tetrasaccharide, or both.

[0024] Next, the present invention provides a composition comprising at least one bacterium selected from (a) to (c) above.

[0025] The composition of the present invention can be a probiotic composition.

[0026] The compositions of the present invention can be used for at least one purpose selected from the group consisting of infants, adults and the elderly.

[0027] The composition of the present invention may be at least one composition selected from the group consisting of intestinal composition, food and beverage composition and nutritional composition.

[0028] The compositions of the present invention may further comprise 2'-fucosylated lactose, and any one or both of 3'-sialyl lactose and lactose-N-tetrasaccharide.

[0029] In addition, the present invention provides a prebiotic composition for promoting the proliferation of at least one bacterium selected from (a) to (c) below, wherein the prebiotic composition comprises 2'-fucosylated lactose, and any one or both of 3'-sialyl lactose and lactose-N-tetrasaccharide.

[0030] (a) Bifidobacterium longum infantis subspecies (NITE BP-03068)

[0031] (b) Bifidobacterium bifidum (NITE BP-03058)

[0032] (c) Bifidobacterium bifidum (NITE BP-03067)

[0033] Furthermore, the present invention also provides a method for manufacturing a composition comprising at least one bacterium selected from (a) to (c) below.

[0034] (a) Bifidobacterium longum infantis subspecies (NITE BP-03068)

[0035] (b) Bifidobacterium bifidum (NITE BP-03058)

[0036] (c) Bifidobacterium bifidum (NITE BP-03067)

[0037] The effects of the invention

[0038] This invention provides novel Bifidobacterium bacteria with the ability to assimilate two or more HMOs.

[0039] It should be noted that the effect is not necessarily limited to the one described here, but can also be any effect described in this specification. Detailed Implementation

[0040] The following describes embodiments for carrying out the present invention. It should be noted that the embodiments described below illustrate an example of a representative embodiment of this disclosure and are therefore not intended to narrowly interpret the scope of the invention. It should also be noted that in this specification, when a numerical range is expressed as "lower limit to upper limit," the upper limit can be "below" or "below" the upper limit, and the lower limit can be "above" or "above" the lower limit. Furthermore, percentages in this specification are based on mass unless otherwise stated.

[0041] <Novel Bifidobacteria>

[0042] The novel Bifidobacterium bacteria of the present invention are at least one bacterium selected from (a) to (c) above, capable of assimilating two or more HMOs. Specifically, they possess the assimilatory capacity for any one or both of 2'-fucosylated lactose (hereinafter also referred to as "2'-FL"), 3'-sialyl lactose (hereinafter also referred to as "3'-SL"), and lactose-N-tetrasaccharide (hereinafter also referred to as "LNT"). Therefore, the novel Bifidobacterium bacteria of the present invention can readily proliferate even in the intestine by using 2'-FL, and any one or both of 3'-SL and LNT.

[0043] Bifidobacteria have been reported to have various physiological functions, which are reported to be achieved through proliferation in the intestine and the production of substances (e.g., acetic acid). Therefore, the Bifidobacteria of the present invention also exhibit high safety, and the efficacy commonly attributed to Bifidobacteria can be expected across a wide age range. Therefore, the Bifidobacteria of the present invention can be used in a wide range of compositions (for food products, functional foods, pharmaceuticals, or animal feed, etc.). Furthermore, the Bifidobacteria of the present invention can be expected to have probiotic effects, and therefore can also be used for purposes such as improving health, improving dietary habits, improving the intestinal environment, or preventing / treating intestinal infections. The bacteria are described in detail below.

[0044] (a) Bifidobacterium longum infantis subspecies (NITE BP-03068)

[0045] As shown in the examples described later, Bifidobacterium longum subsp. infantis (NITE BP-03068) exhibits excellent assimilation capacity for 2'-FL, 3'-fucosylated lactose (hereinafter also referred to as "3'-FL"), LNT, and lactose-N-neotetrasaccharide (hereinafter also referred to as "LNnT") in the <Method for Determining the Assimilation Capacity of Sugar Sources> described later, with OD values ​​of ≥0.3 for each.

[0046] Bifidobacterium longum subspecies infantis (NITE BP-03068) is a novel strain of Bifidobacterium longum subspecies infantis. Due to the following bacteriological properties and characteristics, it was internationally deposited on November 20, 2019, as Bifidobacterium infantis (accession number: NITE BP-03068) at the Patent Biocollection Center of the National Institute of Advanced Industrial Science and Technology (National Institute for Technology Evaluation, Patent Microbiology Collection Center (NPMD)) (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kamisamaashi, Kisarazu City, Chiba Prefecture, Japan). This strain is normally available from the aforementioned collection institution. It should be noted that "Bifidobacterium infantis" has been reclassified as "Bifidobacterium longum subspecies infantis," and the two terms have the same meaning.

[0047] (b) Bifidobacterium bifidum (NITE BP-03058)

[0048] As shown in the examples described later, Bifidobacterium bifidum (NITE BP-03058) exhibited excellent assimilation capacity for 2'-FL, 3'-FL, 3'-SL, 6'-sialyl lactose (hereinafter also referred to as "6'-SL"), LNT, and LNnT in the <Method for Determining the Assimilation Capacity of Glycogens> described later, with OD values ​​of ≥0.3.

[0049] Bifidobacterium bifidum (NITE BP-03058) is a novel strain of Bifidobacterium bifidum. Due to the following mycological properties and characteristics, it was internationally deposited as Bifidobacterium bifidum (accession number: NITE BP-03058) on November 8, 2019, at the Patent Biological Collection Center of the National Institute of Advanced Industrial Science and Technology (National Institute for Technology Evaluation, Patent Microbiology Collection Center (NPMD)) (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kamisamaashi, Kisarazu City, Chiba Prefecture, Japan). This strain is normally available from the aforementioned collection institution.

[0050] (c) Bifidobacterium bifidum (NITE BP-03067)

[0051] As shown in the examples described later, Bifidobacterium bifidum (NITE BP-03067) exhibited excellent assimilation capacity for 2'-FL, 3'-FL, 3'-SL, 6'-SL, LNT, and LNnT in the <Method for Determining the Assimilation Capacity of Glycogens> described later, with OD values ​​of ≥0.3.

[0052] Bifidobacterium bifidum (NITE BP-03067) is a novel strain of Bifidobacterium bifidum. Due to the following mycological properties and characteristics, it was internationally deposited as Bifidobacterium bifidum (accession number: NITE BP-03067) at the National Institute of Advanced Industrial Science and Technology (National Institute for Technology Evaluation, Patent Microbiology Collection Center (NPMD)) (Postal Code: 292-0818, Address: Room 122, 2-5-8 Kamisamare, Kisarazu City, Chiba Prefecture, Japan). This strain is normally available from the aforementioned collection institution.

[0053] In this invention, strains substantially homologous to the strains described above may also be used. Strains substantially homologous to the strains of this invention refer to, for example, bacteria of the same genus, i.e., strains whose 16S rRNA gene sequence preferably has at least 99.9% homology, more preferably 100%, to the 16S rRNA gene sequence of these bacteria, and preferably have the same bacteriological properties as these bacteria.

[0054] In this invention, any mutant strain of the aforementioned Bifidobacterium species can be used as long as it possesses characteristics that enable the realization of the invention's objectives (e.g., the ability to assimilate 2'-FL, 3'-SL, and LNT, or both). Furthermore, this mutant strain is preferably a bacterium possessing the same bacteriological properties as the aforementioned Bifidobacterium species and having an assimilation capacity equal to or greater than that of the aforementioned Bifidobacterium species, including 2'-FL, 3'-SL, and LNT, or both. Whether a mutant strain possesses an assimilation capacity "equal to or greater than that of the aforementioned Bifidobacterium species, including 2'-FL, 3'-SL, and LNT, or both," can be confirmed, for example, by the methods described in the embodiments below.

[0055] This mutant strain can also be constructed by non-artificially introducing mutations into the aforementioned Bifidobacterium bacteria. Alternatively, it can be constructed by introducing mutations into the aforementioned bacteria through treatment with mutagens such as UV, or by introducing mutations into the aforementioned strains through various gene manipulation methods.

[0056] It should be noted that the strains specified by the strain names in the above examples are not limited to the strains themselves preserved and registered with the designated institutions under that strain name (hereinafter also referred to as "preserved strains" for ease of explanation), but also include strains that are substantially equivalent to them (also referred to as "derived strains" or "mutated strains") (the same applies to strains hereinafter). Regarding strains, "strains substantially equivalent to the above-mentioned preserved strains" refers to strains belonging to the same species as the above-mentioned preserved strains and capable of obtaining the same or higher-than-ideal characteristics of the present invention as the preserved strain. For example, strains substantially equivalent to the above-mentioned preserved strains can be derived strains from which the preserved strain is used as a parent strain. Examples of derived strains include strains obtained through breeding from the preserved strains and strains naturally produced from the preserved strains.

[0057] The following are examples of strains that are essentially the same strain or derivative strain.

[0058] (1) Strains identified as the same strain by random amplified polymorphic DNA (RAD) or pulsed-field gel electrophoresis (described in Probiotics in food / Health and nutritional properties and guidelines for evaluation 85 Page 43).

[0059] (2) A strain that possesses only genes derived from the preserved strain and no genes derived from foreign sources, with a DNA homology of 95% or higher (preferably 98% or higher).

[0060] (3) Strains obtained from this strain through breeding (including genetic engineering alterations, mutations, or natural mutations) and possessing the same traits.

[0061] <Methods for determining the assimilation of glycogen>

[0062] Each strain was inoculated at 1 v / v% in 200 μL of MRS (de Man-Rogosa-Sharpe) liquid medium containing a sugar source and cultured anaerobically at 37°C. After 16 hours of culture, the turbidity (OD600) was measured and subtracted from the turbidity of a control cultured in the same manner but without inoculation. The difference was used to determine the presence and degree of assimilation according to the following criteria: OD600 of 0.3 or higher was defined as "good assimilation," 0.4 or higher as "better assimilation," 0.5 or higher as "further better assimilation," and 0.6 or higher as "very good assimilation."

[0063] The strains of the present invention can be multiplied, for example, by culturing the strains.

[0064] The cultivation method is not particularly limited as long as the Bifidobacterium spp. of the present invention can proliferate. Methods commonly used in the cultivation of Bifidobacterium spp. can be used with appropriate modifications as needed. For example, the cultivation temperature can be 30–50°C, preferably 35–45°C. Furthermore, cultivation is preferably carried out under anaerobic conditions, for example, by bubbling anaerobic gas such as carbon dioxide. Alternatively, cultivation can be carried out under microaerophilic conditions such as liquid static culture.

[0065] There are no particular limitations on the culture medium used to cultivate the Bifidobacterium spp. of the present invention for proliferation. Culture media commonly used in the cultivation of Bifidobacterium spp. can be used, with appropriate modifications as needed. Specifically, as a carbon source other than various HMOs, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch hydrolysate, or molasses can be used, depending on their assimilability. As a nitrogen source, ammonium salts or nitrates such as ammonia, ammonium sulfate, ammonium chloride, or ammonium nitrate can be used, for example. In addition, as inorganic salts, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, or ferrous sulfate can be used, for example. Furthermore, organic components such as peptone, soybean flour, defatted soybean meal, meat extract, or yeast extract can also be used. Additionally, as a prepared culture medium, MRS medium can be used, for example.

[0066] Preferred assimilatory components of the Bifidobacterium genus of the present invention include 2'-FL, 3'-SL, and LNT, with at least 2'-FL being preferred, and more preferably containing either or both of 3'-SL and LNT, depending on the assimilatory capacity of each strain. Furthermore, various HMOs such as 3'-FL, 6'-SL, and LNnT may also be included, depending on the assimilatory capacity of each strain.

[0067] The 2'-FL used in this invention has the proliferation-promoting effect of the Bifidobacterium spp. of this invention. Furthermore, by combining 2'-FL with either or both of 3'-SL and LNT, depending on the assimilation capacity of each strain, the proliferation-promoting effect of the Bifidobacterium spp. of this invention can be exerted more stably. Further, by combining various HMOs such as 3'-FL, 6'-SL, and LNnT according to the assimilation capacity of each strain, the proliferation-promoting effect of the Bifidobacterium spp. of this invention can be further stabilized.

[0068] The various HMOs used as assimilating components in this invention can be commercially available or prepared from milk, obtained through known methods such as organic synthesis or enzymatic treatment. Alternatively, milk containing various HMOs (e.g., breast milk, milk powder, cow's milk, or dairy products) can also be used. Furthermore, compositions in which various HMOs are blended into milk (e.g., cow's milk, milk powder, or infant formula) (i.e., milk containing HMOs) can also be used.

[0069] In this invention, HMOs that can be used as assimilating components preferably contain 2'-FL, and more preferably contain either or both of 3'-SL and LNT, depending on the assimilative capacity of each strain. Furthermore, depending on the assimilative capacity of each strain, it is even more preferable to contain 3'-FL, 6'-SL, and LNnT. Besides these various HMOs, examples of common HMOs include, for instance, 3-difucosyllactose, 3-fucosyl-3'-sialotrolose, lactose-N-fucopentose I, lactose-N-fucopentose II, lactose-N-fucopentose III, lactose-N-fucopentose V, lactose-N-difucohexasose I, lactose-N-difucohexasose II, lactose-N-sialotrolose, LSTTa, LSTb, and LSTc. The oligosaccharides of the above-mentioned HMOs can be obtained by known manufacturing methods. In this invention, at least one human milk oligosaccharide selected from the group consisting of these components may also be used.

[0070] <Prebiotic for Proliferating Bifidobacteria of the Present Invention>

[0071] The aforementioned 2'-FL, and any one or both of 3'-SL and LNT have a proliferation-promoting effect on the Bifidobacterium bacteria shown in (a) to (c) above, and can be used as prebiotics. Furthermore, depending on the assimilation capacity of each strain, various HMOs such as 3'-FL, 6'-SL, and LNnT can be used alone or in combination of two or more. In addition, any ingredient suitable for various applications can be used and manufactured using known manufacturing methods suitable for various applications.

[0072] The amount or content of various HMOs such as 2'-FL, 3'-SL, LNT, 3'-FL, 6'-SL and LNnT in the prebiotic composition of the present invention is preferably 1 to 1,000,000 parts by weight, more preferably 10 to 10,000 parts by weight, relative to 100 parts by weight of each Bifidobacterium bacteria shown in (a) to (c) above.

[0073] Through the use of this prebiotic, one can also expect the beneficial probiotic effects on human health, such as the intestinal regulating effect, mineral absorption promotion effect, or prevention and improvement effect on inflammatory bowel disease, as shown in (a) to (c) above.

[0074] <Compositions containing Bifidobacterium bacteria of the present invention>

[0075] The compositions of the present invention refer to compositions containing probiotics, food and beverage compositions, and pharmaceutical compositions, etc. Furthermore, the compositions of the present invention are preferably probiotic compositions.

[0076] Compositions containing the various Bifidobacterium species shown in (a) to (c) above can also be used for humans or non-human animals (preferably mammals), more preferably humans or pets, and more preferably humans. Furthermore, the target population of this invention is not particularly limited as long as it is anyone who desires the effects of probiotics, and examples include infants, children, adults, healthy individuals, middle-aged and elderly people, the very elderly, or those with poor intestinal environments. Preferably, this invention is used for at least one application selected from the group consisting of infants, adults, and the elderly.

[0077] Furthermore, the Bifidobacterium bacteria shown in (a) to (c) above used in this invention have fewer side effects and higher safety because they are derived from humans, and therefore can be continuously ingested for extended periods. They can also be used in a wide range of products, including pharmaceuticals and food products. Moreover, the compositions of this invention can be effectively used to prevent, improve, or treat symptoms or diseases through probiotics.

[0078] Thus, the Bifidobacterium bacteria shown in (a) to (c) above can also be contained as active ingredients in probiotic compositions. Furthermore, the Bifidobacterium bacteria shown in (a) to (c) above can also be used in the manufacture of these various preparations or compositions. Moreover, the Bifidobacterium bacteria shown in (a) to (c) above can also be used as bacteria for probiotics.

[0079] The Bifidobacterium bacteria of the present invention can be used directly. Alternatively, the Bifidobacterium bacteria of the present invention can also be used in combination with physiologically, pharmaceutically, or food-grade carriers or diluents.

[0080] The administration or intake of each of the Bifidobacterium species shown in (a) to (c) above is preferably for at least 1 week, more preferably for at least 4 weeks, and even more preferably for daily intake.

[0081] The dosage of each Bifidobacterium species shown in (a) to (c) above is not particularly limited due to its high safety profile; for example, 1 × 10⁻⁶ is preferred. 6 ~1×10 12 CFU / kg body weight / day, more 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. Furthermore, the preferred dosage per individual (body weight) is 10... 7 ~10 14 CFU / day, preferably 10 8 ~10 13 CFU / day, further optimized to 10 9 ~10 12 CFU / day. It should be noted that in this invention, CFU refers to Colony Forming Unit. When the bacteria mentioned above are dead bacteria, CFU can be replaced with cells.

[0082] In addition, the dosage of each Bifidobacterium spp. shown in (a) to (c) above is preferably 0.01 to 100 mL / kg body weight / day, more preferably 0.1 to 10 mL / kg body weight / day.

[0083] <Pharmaceutical Compositions>

[0084] The pharmaceutical composition of the present invention is not particularly limited as long as it contains the Bifidobacterium genus bacteria shown in (a) to (c) above. This pharmaceutical composition can also be used as an intestinal regulating composition, etc., containing the Bifidobacterium genus bacteria shown in (a) to (c) above.

[0085] As a pharmaceutical composition of the present invention, the various Bifidobacterium bacteria shown in (a) to (c) above can be used directly, or they can be mixed with physiologically permissible liquid or solid formulation carriers and formulated for use.

[0086] Furthermore, the pharmaceutical composition of the present invention, as an oral component, uses the Bifidobacterium spp. bacteria shown in (a) to (c) above, which can be obtained from the human intestine, as the active ingredient; therefore, it can be safely administered even to patients suffering from various diseases. In addition, Bifidobacterium spp. bacteria are also present in the intestines of animals; therefore, the present invention is expected to be less likely to produce side effects even with prolonged continuous administration. Furthermore, Bifidobacterium spp. bacteria can be safely administered to infants and children. Therefore, the present invention is also suitable for the prevention, improvement, and / or treatment of diseases or their symptoms in infants and children.

[0087] This invention can be used for therapeutic purposes or for non-therapeutic purposes.

[0088] "Non-therapeutic purposes" refers to the concept excluding medical procedures, i.e., actions performed on the human body for therapeutic purposes. Examples include health enhancement and cosmetic procedures.

[0089] "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 exacerbation of a disease or symptom.

[0090] "Prevention" refers to preventing or delaying the occurrence of disease or symptoms in the target population, or reducing the risk of disease or symptoms in the target population.

[0091] The dosage form of the pharmaceutical compositions of the present invention is not particularly limited. Specifically, examples include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, syrups, suppositories, injections, ointments, patches, eye drops, and nasal drops. Furthermore, during formulation, additives commonly used as formulation carriers, such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring and odor-correcting agents, diluents, surfactants, or solvents for injections, can be used.

[0092] Furthermore, during formulation, the pharmaceutical compositions of the present invention may use excipients, pH adjusters, colorants, and flavoring agents commonly used in formulation. Additionally, provided that the effects of the present invention are not impaired, the pharmaceutical compositions of the present invention may also use known or potentially emerging ingredients that have preventative, ameliorative, and / or therapeutic effects on diseases or symptoms related to the present invention.

[0093] Furthermore, formulation can be carried out using appropriate and well-known methods depending on the dosage form. During formulation, a suitable formulation carrier can also be appropriately mixed in.

[0094] The content of each Bifidobacterium species shown in (a) to (c) above in the pharmaceutical composition of the present invention can be appropriately set according to the dosage form, administration, patient's age, gender, type of disease, severity of disease, and other conditions, and is generally preferably 1 × 10⁻⁶. 6 ~1×10 12 cfu / g or 1×10 6 ~1×10 12 In the range of cfu / mL, more preferably in the range of 1×10⁻⁶. 7 ~1×10 11 cfu / g or 1×10 7 ~1×10 11 Within the range of cfu / mL. In the case that the Bifidobacterium bacteria shown in (a) to (c) above are dead bacteria, cfu / g or cfu / mL can be replaced with cells / g or cells / mL.

[0095] The timing of administration of the pharmaceutical composition of the present invention is not particularly limited, and an appropriate timing of administration can be selected according to the treatment method for the symptoms or disease of the target patient. Furthermore, it can be administered prophylactically or as maintenance therapy. Additionally, the administration method is preferably determined based on the formulation form, the patient's age, sex, other conditions, or the severity of the patient's symptoms. It should be noted that the pharmaceutical composition of the present invention can be administered once daily or in multiple divided doses under any circumstances, and can also be administered once every few days or weeks.

[0096] <Food and beverage composition>

[0097] The food and beverage composition of the present invention can be manufactured by adding the various Bifidobacterium bacteria shown in (a) to (c) above to a known food and beverage, or by mixing the various Bifidobacterium bacteria shown in (a) to (c) above into the raw materials of the food and beverage to form a new food and beverage composition.

[0098] The food and beverage compositions of the present invention are not particularly limited as long as they contain the Bifidobacterium genus bacteria shown in (a) to (c) above. Examples of food and beverage compositions include beverages such as soft drinks, carbonated drinks, nutritional drinks, fruit juice drinks, or lactic acid bacteria drinks (including concentrated stock solutions and powders for preparation of these beverages); frozen desserts such as ice cream, fruit juice ice cream, or shaved ice; confectionery such as maltose, candy, chewing gum, chocolate, compressed candy, snacks, biscuits, jelly, jam, cream, or baked goods; dairy products such as processed milk, milk drinks, fermented milk, lactic acid bacteria drinks, or butter; bread; enteral nutrition foods or liquid foods such as porridge, weaning foods, infant formula, or sports drinks; and other functional foods. In addition, the food and beverage composition can be a supplement, for example, it can also be a tablet supplement. In the case of a supplement, the intake of the Bifidobacterium genus bacteria shown in (a) to (c) above can be used to determine the daily dietary intake and calorie intake without being affected by other foods.

[0099] Furthermore, the food and beverage compositions of the present invention may use ingredients having the aforementioned probiotic effects or ingredients that assist in the probiotic effects, provided that the effects of the present invention are not impaired. For example, the food and beverage compositions of the present invention may be prepared by combining various proteins such as whey protein, casein, soy protein, or pea protein, or mixtures thereof, or their decomposition products; amino acids such as leucine, valine, isoleucine, or glutamine; vitamins such as vitamin B6 or vitamin C; creatine; citric acid; or fish oil, etc., with the various Bifidobacterium bacteria shown in (a) to (c) above.

[0100] Furthermore, the dietary composition defined in this invention can also be provided / sold as a dietary product with indicated uses (including health care uses) such as probiotics. Additionally, as a target audience for the dietary product, it can be provided / sold as "those who wish to live with Bifidobacteria," "those who wish to improve their intestinal environment," "those who wish to adjust their abdominal condition," or "those who wish to establish a healthy intestinal environment," etc.

[0101] The term "marking" includes all actions used to notify the user of the aforementioned purpose. Any action that can evoke / analyze the aforementioned purpose, regardless of the purpose, content, object / medium, etc., falls under the category of "marking" in this invention.

[0102] Furthermore, "labeling" is preferably carried out through means that allow consumers to directly identify the aforementioned uses. Specifically, examples include: items that display the aforementioned uses on food or beverage products or their packaging for the purpose of transfer, delivery, or transfer or delivery; the description and display of the aforementioned uses in import activities, advertisements, price lists, or instruction manuals related to the products, or the act of providing information containing the aforementioned uses using electromagnetic (network, etc.) methods.

[0103] On the other hand, as the labeling content, it is preferable to use labels that are licensed by administrative authorities or other permits (e.g., labels that are licensed under various administrative regulations and are displayed under such permits). Furthermore, it is preferable to attach such labeling content to packaging, containers, catalogs, brochures, or other promotional materials and other materials displayed at the point of sale, such as POP (point of purchase advertising).

[0104] In addition, "labeling" can also include labels for health foods, functional foods, enteral nutrition foods, foods for special purposes, health function foods, foods for specific health uses, nutritional function foods, functionally labeled foods, or quasi-medicines. Among these, labels permitted by the Consumer Affairs Agency are particularly noteworthy, including those permitted under the Foods for Specific Health Uses System, the Nutritional Function Food System, the Functionally Labelled Food System, or similar systems. Specifically, this includes labels for foods for specific health uses, labels for conditionally designated foods for specific health uses, labels indicating effects on the body's structure and function, labels indicating reduced disease risk, or scientifically based functional labels. More specifically, typical examples include labels for foods for specific health uses (especially health-related labels) and similar labels as stipulated in the Cabinet Office Ordinance (Cabinet Office Ordinance No. 57, August 31, 2008) related to the permission for labels for specific uses as stipulated in the Health Promotion Law.

[0105] The content of each Bifidobacterium species shown in (a) to (c) above in the food and beverage composition of the present invention can be appropriately set according to the form of the food and beverage composition, and is generally preferably 1 × 10 in the food and beverage. 6 ~1×10 12 cfu / g or 1×10 6 ~1×10 12 In the range of cfu / mL, more preferably in the range of 1×10⁻⁶. 7 ~1×10 11 cfu / g or 1×10 7 ~1×10 11 Within the range of cfu / mL.

[0106] The dietary composition of the present invention can be used as a nutritional composition. In this invention, "nutritional composition" refers to a dietary composition ingested orally. The types of nutritional compositions that can be used in this invention are not particularly limited, but are preferably formula milk or liquid foods, more preferably formula milk. It can be ingested by infants, toddlers, children, and adults, but is preferably by infants and toddlers.

[0107] Formula milk includes formula milk powder and formula liquid milk.

[0108] In the relevant provincial ordinances on milk and dairy product composition standards (Milk and Dairy Products Ordinance), formula milk powder is defined as "milk powder made from raw milk, cow's milk, special cow's milk, or food made from them, or from which essential nutrients for infants and young children are added, or as the main ingredient."

[0109] In the aforementioned provincial ordinance, formula liquid milk is defined as "liquid milk made by processing raw milk, cow's milk, special cow's milk, or food made from them, or by adding essential nutrients for infants and young children as the main ingredient."

[0110] In addition, formula milk includes various nutrients such as proteins, fats, carbohydrates, minerals or vitamins, and also includes those processed into powder or liquid form.

[0111] In addition, formula milk also includes "infant formula milk powder", "infant formula liquid milk" and "milk powder for pregnant and lactating women" as special purpose foods as stipulated by the Health Promotion Law. It also includes infant formula milk powder for infants aged 0 to 12 months, formula milk powder for infants aged 6 to 9 months and older and young children (up to 3 years old), formula milk powder for low birth weight infants (newborns with a birth weight of less than 2500g), various therapeutic milk powders for infants and young children with conditions such as cow's milk allergy or lactose intolerance, formula milk powder for young children, nutritional powder for adults and nutritional powder for the elderly, etc.

[0112] Furthermore, the food and beverage compositions (including nutritional compositions) of the present invention can be applied to health functional foods or patient foods. The health functional food system is designed in view of internal and external trends, and its integration with existing specific health food systems. It targets not only ordinary foods but also foods formulated into tablets or capsules, and includes two types: specific health foods (individually licensed type) and nutritional functional foods (standard specification type).

[0113] <Method for manufacturing the composition>

[0114] The method for manufacturing the composition of the present invention will be described below. In the method for manufacturing the composition of the present invention, the steps of adding each Bifidobacterium bacteria shown in (a) to (c) above can be performed at any stage of the composition manufacturing process.

[0115] As an example of a method for manufacturing a composition, a method for manufacturing a composition including the following steps A and B can be listed.

[0116] Step A: A step of culturing the various Bifidobacterium species shown in (a) to (c) above in a culture medium to obtain a culture or product containing the various Bifidobacterium species shown in (a) to (c) above.

[0117] Step B: The above-mentioned culture or product is dried to obtain microbial powder or dried product.

[0118] The form of the Bifidobacterium bacteria or cultures containing them shown in (a) to (c) above is not particularly limited, and can be any type, whether liquid or solid. From the viewpoint of ease of handling during manufacturing and storage, the form is preferably a dried form (e.g., bacterial powder or dried culture). In addition, as mentioned above, examples of such cultures include bacterial cells isolated from the culture medium, cultures containing bacterial cells, and cultures from which bacterial cells have been removed.

[0119] The drying methods described above are not particularly limited as long as they do not impair the effectiveness of the present invention. Examples include spray drying, boiling sterilization, freeze drying, UHT sterilization, pressure sterilization, high-pressure steam sterilization, dry heat sterilization, flowing steam sterilization, electromagnetic wave sterilization, electron beam sterilization, high-frequency sterilization, radiation sterilization, ultraviolet sterilization, ethylene oxide gas sterilization, hydrogen peroxide plasma sterilization, and chemical sterilization methods (alcohol sterilization, formalin fixation, and water electrolysis). In this invention, freeze drying or spray drying is preferred.

[0120] In addition, the bacterial cells can also be broken down. The broken material can be broken down live bacteria, broken down dead bacteria, or bacteria that have been subjected to heating or freeze-drying after being broken down.

[0121] From the viewpoint of increasing the viability rate, it is preferable to use the above-mentioned culture for freeze drying; from the viewpoint of increasing production efficiency, it is preferable to use the above-mentioned culture for spray drying.

[0122] As an example of a method for manufacturing a composition, a method for manufacturing a composition including at least one of step C or step D can be listed.

[0123] Step C: The step of mixing the various Bifidobacterium bacteria and prebiotics shown in (a) to (c) above.

[0124] Step D: The step of mixing the various Bifidobacterium bacteria shown in (a) to (c) above with the milk components.

[0125] At this point, the Bifidobacterium species shown in (a) to (c) above can also be used in a composition containing the Bifidobacterium species shown in (a) to (c) above. Furthermore, the composition may also contain other Bifidobacterium species and probiotics such as lactic acid bacteria.

[0126] When this prebiotic is mixed in, it can also be used in any step of the manufacturing process.

[0127] In addition, the milk component is preferably in powder form, and more preferably mixed with the bacterial powder of each of the Bifidobacterium species shown in (a) to (c) above.

[0128] As for the aforementioned milk components, there are no particular limitations as long as they do not impair the effects of the present invention. Examples include cow's milk, buffalo milk, goat's milk, goat's milk, mare's milk, skim milk, skim milk concentrate, skim milk powder, concentrated milk, whole milk powder, cream, butter, skim milk, condensed milk, and milk protein, etc. One or more selected from the group consisting of these components can be used. Furthermore, there are no particular limitations on the milk protein; examples include whey protein, casein, and their hydrolysates, etc. One or more selected from the group consisting of these components can be used. Among the milk components, those derived from cow's milk are preferred.

[0129] The hydrolysate described above can be used to produce whey hydrolysate or casein hydrolysate by hydrolyzing the aforementioned milk components (preferably milk proteins). Examples of hydrolysis methods include acid / alkali hydrolysis and enzymatic hydrolysis. Enzymatic hydrolysis is preferred, and the enzyme used is preferably a proteolytic enzyme. Examples of proteolytic enzymes include proteases, trypsin, chymotrypsin, plasmin, pepsin, papain, peptidase, and aminopeptidase. The pH of the hydrolysis reaction can be appropriately adjusted to the optimal pH of the enzyme used, for example, at pH 2 to 6. The temperature of the hydrolysis reaction is not particularly limited, but it is generally preferred to be carried out in the range of 30 to 60°C, and the reaction time is preferably 2 to 10 hours.

[0130] Furthermore, when preparing supplements or tablets, the composition of the present invention may include a step of mixing a culture containing each of the Bifidobacterium species shown in (a) to (c) above and an excipient, and a step of shaping it into the specified shape. Examples of shaping steps include, for example, tableting; and examples of tableting include, for example, compressing a mixture of powders or granules into tablets.

[0131] Furthermore, as an example of a method for manufacturing the composition of the present invention, a method for manufacturing a fermented food (preferably fermented milk) containing each of the Bifidobacterium bacteria shown in (a) to (c) above can be cited.

[0132] The following steps are performed: the above-mentioned bacterial powder obtained by the above manufacturing method is added to the fermented milk raw material, and fermented milk containing the Bifidobacterium genus bacteria shown in (a) to (c) is obtained. Alternatively, the following steps are performed: the above-mentioned bacterial powder is mixed with fermented milk to obtain fermented milk containing the Bifidobacterium genus bacteria shown in (a) to (c) above. The amount of bacteria of the present invention in this composition is adjusted by adjusting the fermentation conditions and mixing amount to achieve a specified amount of bacteria. Thus, fermented food and beverages (preferably fermented milk or fermented products) containing the Bifidobacterium genus bacteria shown in (a) to (c) above can be provided.

[0133] Example

[0134] The present invention will be further described in detail below based on embodiments. It should be noted that the embodiments described below illustrate an example of a representative embodiment of the present invention and are not intended to narrowly interpret the scope of the present invention.

[0135] For four Bifidobacterium strains owned by Morinaga Milk Industry Co., Ltd. (hereinafter referred to as "candidate strains"), MRS (deMan-Rogosa-Sharpe) liquid culture medium (composition: Table 2) with the sugar source changed to the six human milk oligosaccharides (manufactured by Jennewein Biotechnologie GmbH) listed in Table 1 was prepared. Each strain was inoculated at 1 v / v% in 200 μL of the degassed medium after 16 hours and cultured anaerobically at 37°C. The turbidity (OD600) after 16 hours of culture was measured to determine the presence and degree of assimilation. An OD600 of 0.3 or higher was considered "good assimilation," 0.5 or higher was considered "better assimilation," and 0.8 or higher was considered "very good assimilation."

[0136] In addition, *Bifidobacterium breve* (JCM1192) and *Bifidobacterium longum* subsp. infantis (ATCC15697) were used as comparisons. The average values ​​of turbidity (OD660) measured after 16 hours of incubation are shown in Table 1.

[0137] [Table 1]

[0138]

[0139] [Table 2]

[0140] Table 2. MRS Composition

[0141]

[0142] The results above indicate that the assimilation of 2'-FL was higher in all four candidate strains compared to the control group, and other human lactose oligosaccharides were also assimilated using these strains.

[0143] [Manufacturing Example]

[0144] Examples of manufacturing the compositions, pharmaceutical compositions, fermented milk, formula milk powder and food and beverage compositions of the present invention are shown below, but the compositions of the present invention are not limited thereto.

[0145] [Manufacturing Example 1]

[0146] Bifidobacterium longum subsp. infantis (NITE BP-03068) was added to 3 mL of MRS liquid medium and anaerobically cultured at 37°C for 16 hours. The culture was then concentrated and freeze-dried to obtain a freeze-dried bacterial powder (bacterial endosperm). The bacterial endosperm, 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 (NITE BP-03068).

[0147] [Manufacturing Example 2]

[0148] Bifidobacterium bifidum (NITE BP-03058) was added to 3 mL of MRS liquid medium and anaerobically cultured at 37°C for 16 hours. The culture was then concentrated and freeze-dried to obtain a freeze-dried bacterial powder (bacterial endosperm). The bacterial endosperm, dried milk protein concentrate powder (MPC480, manufactured by Fonterra, protein content 80% by mass, casein:whey protein = approximately 8:2), and prebiotics (2'-FL, 3'-SL, 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 bifidum (NITE BP-03058).

[0149] [Manufacturing Example 3]

[0150] Bifidobacterium bifidum (NITE BP-03067) was added to 3 mL of MRS liquid medium and anaerobically cultured at 37°C for 16 hours. The culture was then concentrated and freeze-dried to obtain a freeze-dried powder (bacterial endosperm) containing Bifidobacterium bifidum (NITE BP-03067). Next, prebiotics (2'-FL, 3'-SL, and LNT) and crystalline cellulose were added to a stirred granulator and mixed. Then, purified water was added for granulation, and the granules were dried to obtain a granule (pharmaceutical composition) containing the bacterial fermentation components, prebiotics, and excipients. Thus, a granule containing Bifidobacterium bifidum (NITE BP-03067) can be obtained.

[0151] [Manufacturing Example 4]

[0152] The method for producing fermented milk with added Bifidobacterium bifidum (NITE BP-03067) is as follows.

[0153] First, the milk raw material is mixed with water and other ingredients as needed, preferably homogenized, and then heat-sterilized. Homogenization and heat sterilization can be performed using conventional methods. Lactic acid bacteria starter is added (inoculated) into the heat-sterilized milk preparation, and fermentation is carried out at a specified temperature to obtain a fermented product. Curd is formed through fermentation.

[0154] As a starter culture for lactic acid bacteria, bacteria commonly used in yogurt production, such as *Lactobacillus bulgaricus*, *Lactococcus lactis*, and *Streptococcus thermophilus*, can be used. Once the target pH value is reached, the formed curd is broken up by stirring and cooled to below 10°C to obtain the fermented product. Cooling to below 10°C reduces the activity of the lactic acid bacteria and inhibits acid production.

[0155] Next, the fermented product obtained in the fermentation process is heated to obtain a heated fermented product (heat-treated fermented product). By moderately heating the fermented product, the acid formation caused by lactic acid bacteria in the heated fermented product can be suppressed. As a result, the decrease in pH during subsequent manufacturing processes and / or storage of concentrated fermented milk containing Bifidobacteria can be suppressed, thereby improving the survival rate of Bifidobacteria.

[0156] Next, Bifidobacterium bifidum (NITE BP-03067) and prebiotics (2'-FL, 3'-SL, and LNT) are added to the post-fermentation product obtained in the heat treatment process. The preferred amount of Bifidobacterium bifidum (NITE BP-03067) added relative to the post-fermentation product is 1 × 10⁻⁶. 7 ~1×10 11 CFU / mL, more preferably 1×10⁻⁶ 8 ~1×10 10 CFU / mL. When Bifidobacterium bifidum (NITE BP-03067) is a dead bacterium, CFU / mL can be replaced with cells / mL.

[0157] After heating, Bifidobacterium bifidum (NITE BP-03067) and prebiotics are added to the fermented product, followed by concentration. The concentration process can be carried out using well-known concentration methods, such as centrifugation or membrane separation.

[0158] In the centrifugation process, whey is removed from the concentrate (heated fermented product with added bifidobacteria and prebiotics) to obtain concentrated fermented milk containing bifidobacteria and prebiotics with increased solids concentration.

[0159] Fermented milk containing Bifidobacterium bifidum (NITE BP-03067) as described above can be produced.

[0160] [Manufacturing Example 5]

[0161] The manufacturing method of formula milk powder with added Bifidobacterium longum infant subsp. (NITE BP-03068) is as follows.

[0162] 10 kg of desalted cow's milk whey protein powder (manufactured by MILEI GmbH), 6 kg of cow's milk casein powder (manufactured by Fonterra), 48 kg of lactose (manufactured by MILEI GmbH), 920 g of mineral mixture (manufactured by Tomita Pharmaceutical Co., Ltd.), 32 g of vitamin mixture (manufactured by Tanabe Pharmaceutical Co., Ltd.), 500 g of lactulose (manufactured by Morinaga Milk Industry Co., Ltd.), 500 g of raffinose (manufactured by Nippon Beet Sugar Co., Ltd.), and 900 g of galacto-oligosaccharide syrup (manufactured by Yakult Pharmaceutical Industry Co., Ltd.) were dissolved in 300 kg of warm water and then heated at 90°C for 10 minutes to dissolve. 28 kg of modified fat (manufactured by Taiyo Oil Co., Ltd.) was added and homogenized. Then, sterilization, concentration, and spray drying were performed to prepare approximately 95 kg of formula milk powder. Add 1.8 × 10⁻⁶ spores of Bifidobacterium longum subsp. infantis (NITE BP-03068) diluted in starch. 11Approximately 95 kg of Bifidobacterium / oligosaccharide blended formula milk powder was prepared by dissolving 100 g of the obtained formula milk powder in water to prepare a formula milk preparation with a total solids concentration of 14% (w / v) as the standard formula milk concentration. The number of Bifidobacterium bifidum in the formula milk preparation was 2.7 × 10⁻⁶. 9 CFU / 100ml.

[0163] The gut environment can be improved by consuming or giving formula milk containing Bifidobacterium longum infantis (NITE BP-03068) as described above.

[0164] [Manufacturing Example 6]

[0165] The manufacturing method of intestinal environment improvement food with added Bifidobacterium bifidum (NITE BP-03058) is as follows.

[0166] Bifidobacterium bifidum (NITE BP-03058) was added to 3 mL of MRS liquid culture medium and anaerobically cultured at 37°C for 16 hours. The culture was then concentrated and freeze-dried to obtain a freeze-dried bacterial powder (bacterial powder), thus yielding the composition of this invention. Alternatively, the bacterial powder of this invention can be added to foods containing a large amount of cellulose (vegetables, salads, etc.) for cooking, thereby obtaining the composition of this invention.

[0167] Alternatively, the microbial powder can be ingested before, during, or after meals. Since dietary fiber is inevitably ingested whenever a regular meal is eaten (a meal consisting of a staple food, side dishes, and a main dish), the dried microbial powder product of this invention can be ingested along with meals, and an improvement in the intestinal environment of adults can be expected.

[0168] By having individuals experiencing intestinal problems such as constipation or diarrhea continuously consume the intestinal environment-improving food of this invention, further improvements in the intestinal environment can be expected.

[0169] As described above, the Bifidobacterium spp. of the present invention, which has the assimilation ability of 2'-FL and 3'-SL, or LNT, and the composition containing the Bifidobacterium spp., and the composition for promoting the proliferation of Bifidobacterium spp., can help promote the proliferation of Bifidobacterium spp. in the intestine. In addition, it can be effectively used for the formation of a good intestinal flora and the improvement of the intestinal environment.

Claims

1. A bacterium selected from (a) to (c) below, (a) Bifidobacterium longum infantis subspecies (NITE BP-03068) (b) Bifidobacterium bifidum (NITE BP-03058) (c) Bifidobacterium bifidum (NITE BP-03067).

2. The bacteria according to claim 1, which has 2'-Fucose-based lactose, and Either or both of 3'-sialyl lactose and lactose-N-tetrasaccharides Assimilation ability.

3. A composition comprising the bacteria of claim 1 or 2.

4. The composition according to claim 3, wherein, The composition is a probiotic composition.

5. The composition according to claim 3 or 4, wherein, The composition is intended for at least one use selected from the group consisting of infants, adults and the elderly.

6. The composition according to any one of claims 3 to 5, wherein, The composition is at least one composition selected from the group consisting of intestinal compositions, food and beverage compositions, and nutritional compositions.

7. The composition according to any one of claims 3 to 6, further comprising: 2'-Fucose-based lactose, and Either or both of 3'-sialyl lactose and lactose-N-tetrasaccharide.

8. A prebiotic composition for promoting the proliferation of bacteria selected from (a) to (c) below, The prebiotic composition contains 2'-Fucose-based lactose, and 3'-Sialolose and lactose-N-tetrasaccharide, or both. (a) Bifidobacterium longum infantis subspecies (NITE BP-03068) (b) Bifidobacterium bifidum (NITE BP-03058) (c) Bifidobacterium bifidum (NITE BP-03067).

9. A method for manufacturing a composition comprising bacteria selected from (a) to (c) below, (a) Bifidobacterium longum infantis subspecies (NITE BP-03068) (b) Bifidobacterium bifidum (NITE BP-03058) (c) Bifidobacterium bifidum (NITE BP-03067).

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