Method for promoting growth of Bifidobacterium bacteria

Abstract

The present invention provides a method for promoting the growth of Bifidobacterium bacteria. The method for promoting the growth of Bifidobacterium bacteria of the present invention is characterized in that a milk culture medium containing one or more selected from cysteine, cystine and salts thereof is heated and then the Bifidobacterium bacteria are cultured in the milk culture medium.

Description

Technical Field

[0001] The present invention relates to a method for promoting the proliferation of Bifidobacterium bacteria. Background Art

[0002] Cysteine ​​is a non-essential amino acid that naturally forms proteins in the form of L-cysteine ​​and is biosynthesized from methionine in the human body. L-cysteine ​​has antioxidant effects, melanin production inhibition effects, and detoxification effects on acetaldehyde produced by alcohol metabolism. It is used in the fields of food, cosmetics, and pharmaceuticals for its whitening and hangover relief effects.

[0003] Cystine is an amino acid formed by oxidizing two molecules of cysteine ​​and connecting them via a disulfide bond. Cystine is poorly soluble in water and is easily reduced to cysteine. It naturally constitutes proteins in the form of L-cystine, and is particularly contained in large quantities in keratin in hair, nails, etc.

[0004] It has been known that cysteine ​​or cystine is added to a culture medium for the proliferation of bifidobacterium bacteria (Non-Patent Document 1). They are essential amino acids for the proliferation of bifidobacterium bacteria. Since cysteine ​​reduces the redox potential, it is considered to be a component suitable for the proliferation of bifidobacterium bacteria that are sensitive to oxygen and hydrogen peroxide. In addition, it is reported that cysteine ​​is added to milk to reduce the redox potential and improve the survival of bifidobacterium during storage (Non-Patent Document 2). However, the purpose of adding cysteine ​​or cystine to a culture medium for the proliferation of bifidobacterium bacteria is only to supplement amino acids and reduce the redox potential.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Rajiv I. Dave & Nagendra P. Shah, International Dairy Journal, Volume 7, Issues 8-9, 1997, pp. 537-545.

[0008] Non-patent document 2: Marie-Pierre Bolduc, et al., International Dairy Journal, Volume 16, Issue 9, 2006, pp. 1038-1048. Summary of the invention

[0009] An object of the present invention is to provide a method capable of simply and efficiently promoting the growth of Bifidobacterium bacteria.

[0010] The present inventors have conducted various studies in view of the above problems, and as a result, found that when Bifidobacterium bacteria are cultured in a milk medium, if the milk medium is made to contain cysteine, cystine or a salt thereof and then heated, and then the Bifidobacterium bacteria are cultured in the milk medium, the growth of the bacteria is promoted compared to the case where the milk medium and cysteine, cystine or a salt thereof are separately heated and then mixed. The reason for this is believed to be that the substance generated by the reaction of the milk medium components with cysteine, cystine or a salt thereof by the simultaneous heating of the milk medium and cysteine, cystine or a salt thereof acts as a growth promoter for Bifidobacterium bacteria. Therefore, the present inventors conducted various studies to search for the above-mentioned growth promoter, and as a result, found that a component with a molecular weight of less than 3000 was separated from a heat-treated product of a milk culture medium containing cysteine, cystine or a salt thereof, the component was retained on an anion exchange resin at a pH of 8.0 or higher, and further eluted from the anion exchange resin in the presence of anions at a specific concentration to obtain a milk culture medium component, and that the milk culture medium component exhibited a growth promoting effect on bacteria of the genus Bifidobacterium. Based on these findings, the present inventors have completed the present invention.

[0011] That is, the present invention provides the following [1] to

[13] .

[0012] [1] A method for promoting the growth of Bifidobacterium bacteria, comprising heating a milk culture medium containing one or more selected from cysteine, cystine and salts thereof, and then culturing Bifidobacterium bacteria in the milk culture medium.

[0013] [2] The method according to [1], wherein the heat treatment is performed at 60 to 125° C. for 10 to 40 minutes.

[0014] [3] The method according to [1] or [2], wherein the milk culture medium contains one or more selected from the group consisting of L-cysteine, L-cystine, and salts thereof.

[0015] [4] The method according to any one of [1] to [3], wherein the milk culture medium contains L-cysteine ​​hydrochloride.

[0016] [5] A method for producing fermented milk, comprising heating a milk culture medium containing one or more selected from cysteine, cystine and salts thereof, and then culturing Bifidobacterium bacteria in the milk culture medium.

[0017] [6] A fermented milk obtained by the method of [5].

[0018] [7] A growth promoter for Bifidobacterium bacteria, comprising as an active ingredient a heat-treated product of a milk culture medium containing one or more species selected from cysteine, cystine, and salts thereof.

[0019] [8] The growth promoter according to [7], wherein the heat treatment is performed at 60 to 125° C. for 10 to 40 minutes.

[0020] [9] The growth promoter according to [7] or [8], wherein the milk culture medium contains one or more selected from L-cysteine, L-cystine, and salts thereof.

[0021]

[10] The growth promoter according to any one of [7] to [9], wherein the milk culture medium contains L-cysteine ​​hydrochloride.

[0022]

[11] A growth promoter according to any one of [7] to

[10] , wherein the heat-treated product is a component of a heat-treated milk culture medium having a molecular weight of less than 3000, which is maintained on an anion exchange resin at a pH of 8.0 or above and eluted from the anion exchange resin in the presence of 1 to 400 mM anions.

[0023]

[12] A milk culture medium component, which is a component having a molecular weight of less than 3000 in a heat-treated milk culture medium containing one or more selected from cysteine, cystine and their salts, which is maintained on an anion exchange resin at a pH of 8.0 or above and eluted from the anion exchange resin in the presence of 1 to 400 mM anions.

[0024]

[13] A method for producing a milk culture medium component, characterized in that a milk culture medium containing one or more selected from cysteine, cystine and their salts is heat-treated, and components with a molecular weight of less than 3000 are separated from the obtained heat-treated milk culture medium, and the obtained components are retained on an anion resin at a pH of 8.0 or above, and further eluted from the above-mentioned anion resin in the presence of 1 to 400 mM anions.

[0025] According to the present invention, by heating a milk culture medium containing one or more selected from cysteine, cystine and their salts, and then culturing Bifidobacterium bacteria with the milk culture medium, the proliferation of Bifidobacterium bacteria can be simply and efficiently promoted, and the culture time can be shortened. The method for promoting the proliferation of Bifidobacterium bacteria is also useful as a method for producing fermented milk. In addition, the components separated from the heat-treated product of the milk culture medium containing one or more selected from cysteine, cystine and their salts under specific conditions are more useful as a proliferation promoter for Bifidobacterium bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The graph shows the proliferation of Lactobacillus bacteria strains and Bifidobacterium bacteria strains when milk and L-cystine or L-cysteine ​​hydrochloride are heated simultaneously or separately.

[0027] Figure 2 This is a graph showing the growth curve of Bifidobacterium bacterial strains when milk and L-cystine or L-cysteine ​​hydrochloride are heated simultaneously or separately.

[0028] Figure 3 The graph shows the difference in culture properties of Bifidobacterium breve YIT12272 (BbY) due to the difference in the concentration of L-cystine or L-cysteine ​​hydrochloride added to the skim milk medium.

[0029] Figure 4 This is a graph showing the differences in culture properties of BbY due to differences in the concentrations of L-cystine or L-cysteine ​​hydrochloride added to a whey medium.

[0030] Figure 5 The graph shows the difference in culture properties of Bifidobacterium bifidum YIT10347 (BF-1) due to the difference in the concentration of L-cystine or L-cysteine ​​hydrochloride added to the skim milk medium.

[0031] Figure 6 This is a graph showing the differences in culture properties of BF-1 due to differences in the concentrations of L-cystine or L-cysteine ​​hydrochloride added to the whey medium.

[0032] Figure 7 This is a graph showing the culture characteristics of BbY at the L-cystine or L-cysteine ​​hydrochloride concentration at which the maximum viable cell count was observed.

[0033] Figure 8 This is a graph showing the culture characteristics of BF-1 at the L-cystine or L-cysteine ​​hydrochloride concentration at which the maximum number of viable cells was observed.

[0034] Fig. 9 The graph shows the viable counts of each fraction for the samples heated simultaneously (upper section) and the samples heated separately (lower section). The vertical axis shows the ratio of the viable counts of each fraction when the viable count of the control sample is set to 1, and the horizontal axis shows the elution volume.

[0035] Fig.10 The graph shows the acidity of each fraction for samples heated simultaneously (upper row) and samples heated separately (lower row). The vertical axis shows the difference in acidity of each fraction when the acidity of the control sample is set to 0, and the horizontal axis shows the elution volume. DETAILED DESCRIPTION

[0036] The method for promoting growth of Bifidobacterium bacteria of the present invention is characterized by heating a milk culture medium containing one or more selected from cysteine, cystine and salts thereof, and then culturing Bifidobacterium bacteria in the milk culture medium.

[0037] The type of Bifidobacterium bacteria used in the present invention is not particularly limited. For example, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium animalis, Bifidobacterium suis, Bifidobacterium infantis, Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, Bifidobacterium lactis, Bifidobacterium globosum, etc. may be mentioned. Among them, Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium bifidum are preferred because they have been used in dairy products and have accumulated safety data. Bifidobacterium breve and Bifidobacterium bifidum are more preferred. Specifically, Bifidobacterium breve YIT 12272 (FERM BP-11320) and Bifidobacterium bifidum YIT10347 (FERM BP-10613) are particularly preferred.

[0038] The culture medium used in the present invention for the proliferation of Bifidobacterium bacteria is a milk culture medium. Here, the milk culture medium is not particularly limited as long as it is a culture medium with milk as the main component. As milk, milk (whole milk), skim milk as its processed product, whey with casein removed, components from whey, peptides from milk, etc. can be mentioned. As a component from whey, it is preferably a water-soluble protein (lactalbumin) contained in whey, specifically, β-lactoglobulin, α-lactalbumin, etc. can be exemplified. As a milk culture medium, skim milk culture medium and whey culture medium are preferred. Depending on the milk raw material used and its blending amount, the fat-free milk solid component and fat component can be arbitrarily set. Growth factors of Bifidobacterium bacteria such as yeast extract can also be added to the milk culture medium.

[0039] As the cysteine ​​or cystine used in the present invention, it can be cysteine ​​or cystine of natural origin, or it can be cysteine ​​or cystine produced by chemical synthesis, fermentation or gene recombination. As cysteine ​​or cystine, any one of L-body, D-body and DL-body can be used, preferably L-body. As the salt of cysteine ​​or cystine, as long as it is a pharmacologically allowed salt, for example, salts with alkali metals (sodium, potassium, etc.), salts with alkaline earth metals (calcium, magnesium, etc.), ammonium salts, salts with inorganic acids (hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.) and salts with organic acids (acetic acid, propionic acid, tartaric acid, fumaric acid, maleic acid, succinic acid, malic acid, citric acid, etc.). Among them, from the viewpoint of safety and ease of operation, L-cysteine ​​hydrochloride and L-cystine as food additives are preferably used, and L-cysteine ​​hydrochloride is more preferably used. Cysteine, cystine or their salts can be used alone, respectively, or two or more can be used in combination.

[0040] The content of one or more selected from cysteine, cystine and salts thereof (hereinafter referred to as cysteine ​​etc.) in the milk medium is not particularly limited, but is preferably 0.001 to 0.1 (w / v)%, more preferably 0.001 to 0.05 (w / v)%, and further preferably 0.005 to 0.05 (w / v)% in terms of the final concentration from the viewpoint of the proliferation promoting effect. Cysteine ​​etc. is added to the milk medium before the heat treatment. The addition method is not particularly limited, and cysteine ​​etc. may be directly added to the milk medium in such a manner as to achieve the above-mentioned final concentration, or a solution of cysteine ​​etc. (e.g., an aqueous solution) may be added to the milk medium.

[0041] In the method for promoting the proliferation of bifidobacterium bacteria of the present invention, a milk culture medium containing cysteine ​​and the like is subjected to heat treatment. The apparatus for heat treatment is not particularly limited, and examples thereof include an autoclave and the like. The heating temperature is preferably 60 to 125°C, more preferably 64 to 121°C, further preferably 100 to 121°C, and further preferably 115 to 121°C. The heating time is preferably 10 to 40 minutes, more preferably 15 to 30 minutes, and further preferably 20 to 30 minutes. Alternatively, the heat treatment may be performed in a manner that satisfies a thermal history (the total amount of heat applied) or a heat sterilization intensity equivalent to the above-mentioned heat treatment conditions. Regarding the temperature of the heat-treated milk culture medium, it is sufficient to leave it to stand or use a cooling device or the like to reduce it to below the culture temperature until the time of culture.

[0042] Next, the Bifidobacterium bacteria are inoculated in the heat-treated milk culture medium for culture. The culture of the Bifidobacterium bacteria can be directly applied under the usual culture conditions. That is, it is sufficient to appropriately set various conditions such as the inoculation amount, temperature, time, and culture atmosphere of the Bifidobacterium bacteria inoculated in the culture medium. For example, the inoculation amount can be 0.01 to 5%, preferably 0.1 to 1%, the culture temperature can be 25 to 46°C, preferably 35 to 42°C, and the culture time can be 6 to 120 hours, preferably 24 to 72 hours. In addition, the culture atmosphere can be aerobic conditions or anaerobic conditions, preferably anaerobic conditions, and the culture method can be any one of standing, stirring, shaking, etc., without particular restrictions.

[0043] According to the method for promoting growth of Bifidobacterium bacteria of the present invention, Bifidobacterium bacteria can be efficiently grown in a short time using a milk medium, and thus the method is also useful as a method for producing fermented milk containing Bifidobacterium bacteria.

[0044] The culture of the bifidobacterium bacteria obtained by the method of the present invention, i.e., fermented milk, can be made into a final product by adding any ingredients such as syrup (sweetener). Examples of syrup include sugars such as glucose, sucrose, fructose, fructose glucose syrup, glucose fructose syrup, palatinose, trehalose, lactose, xylose, maltose, honey, molasses, etc.; sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, isomalt, reduced maltose, reduced maltose syrup, etc.; and high-intensity sweeteners such as aspartame, thaumatin, sucralose, acesulfame potassium, and stevioside. In addition, fermented milk may be mixed with emulsifiers such as sucrose fatty acid ester, glycerol fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester, lecithin, etc.; thickeners (stabilizers) such as agar, gelatin, carrageenan, guar gum, xanthan gum, pectin, locust bean gum, gellan gum, carboxymethyl cellulose, soybean polysaccharides, propylene glycol alginate, etc. In addition, vitamins such as vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, etc.; minerals such as calcium, magnesium, zinc, iron, manganese, etc.; acidulants such as citric acid, lactic acid, acetic acid, malic acid, tartaric acid, gluconic acid, etc.; milk fats such as cream, butter, sour cream, etc.; flavors such as yogurt, berry, orange, pear, perilla, citrus, apple, mint, grape, apricot, pear, custard, peach, melon, banana, tropical, vanilla, black tea, coffee, etc.; herb extracts, brown sugar extracts, etc.

[0045] In order to manufacture fermented milk products, conventional methods can be used. For example, fermented milk can be homogenized to obtain a fermented milk base, and then a separately prepared syrup solution is added and mixed, and then homogenized by a homogenizer, and then flavorings are added, and finally processed into a final product. Fermented milk products include beverages and foods, and can also be made into any form of products such as original flavor, soft type, fruit flavor, solid, liquid, etc. without syrup (sweetener).

[0046] As shown in the examples described later, if a substance obtained by heat-treating a milk culture medium containing cysteine, etc. is used for culture, the proliferation of bifidobacterium bacteria can be promoted. It is believed that this is because the substance generated by the reaction of the milk culture medium components with cysteine, etc. by the simultaneous heat treatment of the milk culture medium and cysteine, etc., contributes to the proliferation promotion of bifidobacterium bacteria. Therefore, the heat-treated substance of the milk culture medium containing cysteine, etc. can be used as a proliferation promoter for bifidobacterium bacteria. As heat treatment conditions, the above conditions can be cited. It should be noted that since cysteine, etc. are amino acids, it is assumed that a protein or peptide is used as a milk culture medium component that reacts with cysteine, etc.

[0047] As shown in the examples described below, the milk medium component in the heat-treated product of a milk medium containing cysteine ​​and the like that shows an effect of promoting the growth of bacteria of the genus Bifidobacterium is more specifically a component having a molecular weight of less than 3000 contained in the heat-treated product of the above-mentioned milk medium, and is a milk medium component that is maintained on an anion exchange resin at a pH of 8.0 or above and eluted from the anion exchange resin in the presence of 1 to 400 mM, preferably 50 to 200 mM, of anions.

[0048] The above-mentioned milk culture medium component can be produced by the following method: heat-treating a milk culture medium containing cysteine ​​and the like, fractionating the components of the obtained milk culture medium having a molecular weight of less than 3000, retaining the obtained components on an anion exchange resin at a pH of 8.0 or above, and further eluting them from the anion exchange resin in the presence of 1 to 400 mM, preferably 50 to 200 mM, of anions.

[0049] Here, the content of cysteine ​​and the like in the milk culture medium when the heat-treated product is obtained is not particularly limited, but from the viewpoint of production efficiency, the final concentration is preferably 0.001 to 1 (w / v)%, more preferably 0.001 to 0.5 (w / v)%, further preferably 0.001 to 0.1 (w / v)%, further preferably 0.001 to 0.05 (w / v)%, and particularly preferably 0.005 to 0.05 (w / v)%. Cysteine ​​and the like are added to the milk culture medium before the heat treatment. The addition method is not particularly limited, and cysteine ​​and the like may be directly added to the milk culture medium in such a manner as to achieve the above-mentioned final concentration, or a solution of cysteine ​​and the like (e.g., an aqueous solution) may be added to the milk culture medium.

[0050] As the heat treatment conditions, the above conditions can be cited. Specifically, the heating temperature is preferably 60 to 125°C, more preferably 64 to 121°C, further preferably 100 to 121°C, and further preferably 115 to 121°C. The heating time is preferably 10 to 40 minutes, more preferably 15 to 30 minutes, and further preferably 20 to 30 minutes. Alternatively, the heat treatment can be performed in a manner that satisfies the thermal history (the sum of the applied heat) or the heat sterilization intensity equivalent to the above-mentioned heat treatment conditions.

[0051] Next, components with a molecular weight of less than 3000 are separated from the heat-treated product of the obtained milk culture medium. Here, as a method for separating components based on molecular weight, any known method can be used, for example, ultrafiltration, gel filtration chromatography, etc. For example, when ultrafiltration is used, an ultrafiltration filter with a molecular weight cutoff of about 3 kDa can be used to recover the filtrate. The molecular weight of the obtained component can be confirmed by SDS-polyacrylamide gel electrophoresis.

[0052] Next, the obtained component with a molecular weight of less than 3000 is adjusted to pH 8.0 or higher, preferably pH 8.0, with hydrochloric acid or the like, and brought into contact with an anion exchange resin to retain the desired component. The pH adjustment is to make the desired component negatively charged and adsorbed to the anion exchange resin.

[0053] Here, the material, structure, etc. of the anion exchange resin are not particularly limited and can be appropriately selected. As the resin matrix of the anion exchange resin, for example, styrene resins, acrylic resins, agarose resins, etc. can be cited. As the resin structure of the anion exchange resin, for example, gel type, porous type, high pore type can be cited. As the functional group of the anion exchange resin, for example, primary amino group, secondary amino group, tertiary amino group, quaternary ammonium group, etc. can be cited. As the anion exchange resin, it can be a strongly basic anion exchange resin or a weakly basic anion exchange resin. From the viewpoint of separation ability, it is preferably a strongly basic anion exchange resin.

[0054] As such anion exchange resin, commercially available products can also be used. For example, as the strongly basic anion exchange resin, Diaion (registered trademark) SA10A, SA12A, SA11A, NSA100, UBA120, PA306S, PA308, PA312, PA316, PA318L, HPA25, SA20A, SA21A, PA408, PA412, PA418 (manufactured by Mitsubishi Chemical Corporation), Q Sepharose (registered trademark) High Performance, Q Sepharose Fast Flow (manufactured by GE Healthcare Japan Co., Ltd.), etc. can be mentioned. As the weakly basic anion exchange resin, Diaion (registered trademark) WA10, WA20, WA21J, WA30 (manufactured by Mitsubishi Chemical Corporation), DEAE Sepharose Fast Flow (manufactured by GE Healthcare Japan Co., Ltd.), etc. can be mentioned. The anion exchange resin is preferably preliminarily equilibrated with a buffer having a buffer capacity of pH 8.0 (eg, Tris buffer).

[0055] The contact method with the anion exchange resin can be batch or column, and is preferably column-type from the perspective of efficiency. In the case of the column, it is sufficient to pass a solution containing a component with a molecular weight of less than 3000 of the heat-treated milk culture medium obtained above through a column filled with anion exchange resin. Then, a sufficient amount of a buffer solution (such as Tris buffer) with a buffering capacity of pH 8.0 is passed to rinse the substance that is not adsorbed on the anion exchange resin. Here, the column capacity and flow rate are not particularly limited, as long as they are appropriately selected in consideration of the sample amount, the retention performance of the anion exchange resin, etc. For example, as the column capacity, 1 to 20 mL, preferably 5 to 20 mL, can be cited, and as the flow rate, 0.1 to 20 mL / min, preferably 1 to 10 mL / min can be cited. The above conditions can be appropriately changed according to the purification amount.

[0056] As such a column filled with an anion exchange resin, commercially available products can also be used. For example, as a column filled with a strongly basic anion exchange resin, HiTrap (registered trademark) Q FF, HiPrep (registered trademark) Q FF (manufactured by GE HEALTHCARE JAPAN Co., Ltd.) and the like can be cited, and as a column filled with a weakly basic anion exchange resin, HiTrap DEAE FF (manufactured by GE HEALTHCARE JAPAN Co., Ltd.) and the like can be cited.

[0057] The components retained in the anion exchange resin are eluted from the anion exchange resin in the presence of 1 to 400 mM, preferably 50 to 200 mM, of anions. The type of anion is not particularly limited, and examples thereof include Cl - ions, etc. Specifically, as long as a buffer solution (such as Tris buffer) of pH 8.0 containing 1 to 400 mM, preferably 50 to 200 mM NaCl is passed through an anion exchange resin, the eluate is recovered. In the case of elution, the flow rate is not particularly limited, as long as it is appropriately selected considering the retention performance of the anion exchange resin, for example, 0.1 to 20 mL / min, preferably 1 to 10 mL / min. The above conditions can be appropriately changed according to the purification amount. The obtained eluate can be directly added to the culture medium for use, or it can be concentrated, diluted, etc. according to the purpose and then used. The milk culture medium components eluted in this way can promote the proliferation of Bifidobacterium bacteria as shown in the examples described below.

[0058] Hereinafter, a method for producing a milk culture medium component as a growth promoting agent of the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

[0059] A whey medium containing cysteine ​​and the like at a final concentration of 0.001 to 1 (w / v)% is heated at 115°C for 30 minutes. The resulting heat-treated whey medium is passed through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and components with a molecular weight of less than 3000 are recovered as a filtrate. The pH of the recovered filtrate is adjusted to 8.0 by hydrochloric acid. On the other hand, a 20 mM Tris buffer at pH 8.0 is passed through an anion exchange column (e.g., HiTrap Q FF (manufactured by GE HEALTHCARE JAPAN Co., Ltd.)) for equilibration. The pH-adjusted filtrate is passed through an anion exchange column that has been equilibrated to retain the desired components on the anion exchange column. For substances that are not adsorbed on the anion exchange column, a sufficient amount of 20 mM Tris buffer is passed through the anion exchange column for washing. Next, the eluate is recovered by passing a pH 8.0 Tris buffer containing 50 to 200 mM NaCl through the column. In addition, when recovering the desired milk culture medium component from the anion exchange column, when a liquid chromatography apparatus is used, for example, a fraction having an elution volume of 22 to 28 mL may be recovered under the following conditions.

[0060] Column: HiTrap Q FF 1mL

[0061] Starting buffer: 20 mM Tris buffer (pH 8.0)

[0062] Dissolution buffer: 20 mM Tris buffer (pH 8.0) containing 1 M NaCl

[0063] Dissolution method: Flowthrough 20CV, Gradient (0→100%, 20CV), 2mL / fraction, wash (100%, 5CV)

[0064] Addition method: Direct sample load 10mL

[0065] Flow rate: 1mL / min

[0066] Column temperature: room temperature

[0067] Equipment: AKTA detector (GE HEALTHCARE)

[0068] Example

[0069] Next, the present invention will be described in further detail with reference to Examples, but the present invention is not limited to these Examples at all.

[0070] Example 1 Growth-promoting effect of heating milk and cystine or cysteine ​​simultaneously

[0071] (1) Test strains

[0072] Bifidobacterium breve YIT 12272 (hereinafter referred to as BbY), Bifidobacterium bifidum YIT 10347 (hereinafter referred to as BF-1), Lactobacillus casei YIT 9029 (hereinafter referred to as YIT 9029), and Lactobacillus acidophilus YIT 0198 (hereinafter referred to as YIT0198) were used.

[0073] (2) Culture medium preparation

[0074] (i) Skim milk culture medium

[0075] Skim milk powder (ABC; manufactured by Snow Brand Dairy Co., Ltd.) was dissolved in RO water to a concentration of 12% (w / v), and 10 mL of the solution was dispensed into a medium-sized test tube with a rim while replacing the gas phase with nitrogen. The tube was sealed with a butyl rubber stopper. The skim milk medium (no-additive medium) was prepared by heat sterilization at 115°C for 30 minutes in an autoclave (SX-500; manufactured by TOMY Seiko Co., Ltd.). In addition, under anaerobic conditions, skim milk medium was prepared by adding an aqueous suspension of L-cystine (manufactured by Kyowa Hakko Biotech Co., Ltd.) or an aqueous solution of L-cysteine ​​hydrochloride (manufactured by Nikkatsu Kagaku Co., Ltd.) to a final concentration of 0.05 (w / v)% before heat sterilization and then heat sterilizing (simultaneous heating medium) and skim milk medium was prepared by adding an aqueous suspension of L-cystine (manufactured by Kyowa Hakko Biotech Co., Ltd.) or an aqueous solution of L-cysteine ​​hydrochloride (manufactured by Nikkatsu Kagaku Co., Ltd.) to a final concentration of 0.05 (w / v)% after heat sterilization and immediately before inoculation (separate heating medium). It should be noted that the aqueous suspension of L-cystine and the aqueous solution of L-cysteine ​​hydrochloride were prepared as a 5% (w / v) aqueous suspension and an aqueous solution, respectively, and 1% (v / v) was added to each medium. In order to make the changes in sample concentration and redox potential caused by the addition of solutions consistent between samples, 1% (v / v) sterile water was added to the simultaneously heated culture medium just before inoculation, 1% (v / v) sterile water was added to the separately heated culture medium before heating, and 1% (v / v) sterile water was added twice to the non-added culture medium, before heating and just before inoculation.

[0076] (3) Inoculation and culture conditions

[0077] When inoculating the bacteria, the inoculation was performed while the gas phase in the container was replaced with nitrogen, and nitrogen was sealed with a butyl rubber stopper for culturing. The culture was performed in a constant temperature water bath set at 37°C for 12 hours.

[0078] (4) Various parameter measurement methods

[0079] (i) pH

[0080] The pH of the culture medium or bacterial solution was measured using a tabletop pH meter (F52; manufactured by Horiba, Ltd.).

[0081] (ii) Acidity

[0082] The amount of 0.1 N sodium hydroxide aqueous solution per 9.0 g of the bacterial liquid required to achieve a pH of 8.5 was defined as the acidity. An automatic titrator (manufactured by Hiranuma Sangyo Co., Ltd.) was used for the measurement.

[0083] (iii) Number of viable cells

[0084] Regardless of the strain, the bacterial solution was appropriately diluted with a 0.85% (w / v) sodium chloride aqueous solution (physiological saline). In the case of Bifidobacterium bacteria, the diluted bacterial solution was inoculated in TOS propionic acid agar medium (manufactured by Yakult Pharmaceutical Industry Co., Ltd.) using a spiral inoculator EDDY JET (manufactured by IUL Instruments GmbH), and in the case of Lactobacillus bacteria, the diluted bacterial solution was inoculated in a plate counting medium (manufactured by Eiken Chemical Co., Ltd.) supplemented with BCP using a spiral inoculator EDDY JET (manufactured by IUL Instruments GmbH), and after culturing at 37°C for 48 to 72 hours, the resulting colonies were counted as the number of viable cells. In the case of Lactobacillus bacteria, the culture was carried out under aerobic conditions, and in the case of Bifidobacterium bacteria, the culture was carried out under anaerobic conditions (Anaeropack; manufactured by Mitsubishi Gas Chemical Co., Ltd.). The colony count was performed using a colony counter ProtoCOL (manufactured by Synoptics Ltd.).

[0085] (5) Results

[0086] exist Figure 1 The growth properties of each strain when skim milk and L-cystine or L-cysteine ​​hydrochloride (hereinafter referred to as L-cysteine) were heated simultaneously or separately are shown in FIG.

[0087] In the case of two Lactobacillus strains (YIT 0198 and YIT 9029), no promotion of growth was observed by the addition of L-cystine or L-cysteine, and no difference was observed due to the timing of adding L-cysteine.

[0088] On the other hand, in the case of BbY, which is a bifidobacterium, it was confirmed that the acidity increased by 2.2 to 2.6 times and the number of viable bacteria increased by 5.6 times in the 12-hour culture compared with the case without addition of L-cystine. In addition, when L-cystine and milk were heated at the same time, the number of viable bacteria after 12 hours of culture was the same as when they were heated separately, but the acidity was higher at 1.3 mL / 9 g. Thus, under the condition that the culture time is long enough, the number of viable bacteria is in a state where the number is large enough (for example, 10 9 CFU / mL or more), but when the acidity is high, it can be judged that the proliferation is high. This is because the number of viable bacteria on the side with high proliferation will reach the peak first, and then the number of viable bacteria on the side with low proliferation will catch up. Therefore, by independently Figure 1 The proliferation curve of BbY was studied when L-cystine or L-cysteine ​​was added at a final concentration of 0.03% (w / v). Figure 2 As shown, it can be seen that the increase rate of the number of viable bacteria is faster when heating is performed simultaneously, and the increase in acidity also becomes higher.

[0089] In the case of BF-1, which is also a bacterium of the genus Bifidobacterium, it was also confirmed that the acidity increased by 1.1 to 1.5 times and the number of viable bacteria increased by 1.7 to 7.7 times in the 12-hour culture compared to the case without addition of L-cystine. When L-cystine and milk were heated simultaneously, the acidity was 1.3 times higher and the number of viable bacteria was 4.5 times higher than when they were heated separately.

[0090] The same experiment was performed three times for the two strains of Bifidobacterium, and all the results showed the same trend. Therefore, it was shown that the growth promotion effect when skim milk and L-cystine or L-cysteine ​​are heated simultaneously is specific to Bifidobacterium bacteria.

[0091] Even when L-cysteine, which is easily soluble in water, was added instead of L-cystine, similarly to L-cystine, it was confirmed that the proliferation was promoted by heating the milk simultaneously ( Figure 1 , 2 ), thus indicating that the increase in the amount of L-cystine dissolved by heating is not the main cause of the proliferation-promoting effect.

[0092] In addition, the culture properties of BbY were compared between the case where L-cysteine ​​was heated alone and added to milk at a final concentration of 0.03% (w / v) (separate heating) and the case where L-cysteine ​​was not heated but filter-sterilized through a 0.22 μm filter and added to milk at a final concentration of 0.03% (w / v) (filter sterilization). As shown in the following Table 1, no difference was observed. Therefore, the possibility that the growth-promoting effect of L-cysteine ​​is attenuated by heating is ruled out.

[0093] [Table 1]

[0094]

[0095] Example 2 Effect of the added concentration of cystine or cysteine ​​on the culture characteristics of bacteria

[0096] (1) Test strains

[0097] Use BbY and BF-1.

[0098] (2) Culture medium preparation

[0099] (i) Skim milk culture medium

[0100] A heating medium containing milk and L-cystine or L-cysteine ​​was prepared in the same manner as in Example 1. The concentration of L-cystine or L-cysteine ​​added was 0.001, 0.003, 0.005, 0.01, 0.03, 0.05, or 0.1% (w / v) as the final concentration.

[0101] (ii) Separation of whey culture medium before heating

[0102] The pH of 12% (w / v) skim milk prepared by dissolving skim milk powder in RO water to 12% (w / v) was adjusted to 4.6 by 5N HCl and centrifuged at 3,000×g for 5 minutes. Then, the pH of the supernatant was adjusted to 6.5 by 5N NaOH and centrifuged at 10,000×g for 15 to 30 minutes. While replacing the gas phase in the medium-sized test tube with nitrogen, the supernatant was dispensed in 10 mL at a time and sealed with a butyl rubber stopper. Then, it was heat-sterilized at 115°C in an autoclave for 30 minutes to prepare a pre-heating separated whey medium. In addition, under anaerobic conditions, a medium (simultaneous heating medium) was prepared in which L-cystine or L-cysteine ​​was added before heating and then heat-sterilized, as in the case of the skim milk medium. The concentration of L-cystine or L-cysteine ​​added was 0.001, 0.003, 0.005, 0.01, 0.03, 0.05, or 0.1% (w / v) as the final concentration.

[0103] (3) Inoculation and culture conditions

[0104] Inoculation and culturing were carried out in the same manner as in Example 1 except that the culturing time was changed to 9 hours when culturing BbY only in the skim milk medium.

[0105] (4) Various parameter measurement methods

[0106] (i) pH, (ii) acidity, and (iii) viable cell count were measured in the same manner as in Example 1.

[0107] (5) Results

[0108] The acidity and viable count of BbY and BF-1 when the final concentration of L-cystine or L-cysteine ​​added to the skim milk medium or the pre-heated separated whey medium was changed are shown in FIG. Figures 3 to 6 . Figure 3 This shows the situation of culturing BbY in skim milk medium. Figure 4 This shows the situation of culturing BbY in whey medium. Figure 5 This shows the situation of culturing BF-1 in skim milk medium. Figure 6 Figure 2 shows the case of culturing BF-1 in whey medium. Figures 3 to 6However, regarding the viable bacterial count and acidity when neither L-cystine nor L-cysteine ​​was added, when BbY was cultured in skim milk medium, it was 5.67×10 8 CFU / mL and 2.48mL / 9g, and 7.42×10 7 CFU / mL and 1.05mL / 9g, and 3.81×10 in the case of BF-1 cultured in skim milk medium. 8 CFU / mL and 2.53mL / 9g, and 5.59×10 in the case of BF-1 cultured in whey medium. 7 CFU / mL and 1.21mL / 9g.

[0109] Whey is a component obtained by removing casein from skim milk, and contains water-soluble components in skim milk such as whey protein, lactose, and minerals. Whey is separated as a supernatant by adjusting the pH of skim milk to 4.6, and the pH is adjusted to 6.5 before being used for culture.

[0110] As a result of the culture, for both BbY and BF-1, the number of viable cells and the acidity increased when the medium was heated while adding L-cystine or L-cysteine, compared with the case of using a medium to which neither L-cystine nor L-cysteine ​​was added.

[0111] Regarding BbY, the number of viable cells in the culture medium heated at the same time reached the maximum when the L-cystine concentration was 0.01% (w / v) for both the skim milk culture medium and the whey culture medium. In addition, the L-cysteine ​​concentration at which the number of viable cells in the culture medium heated at the same time reached the maximum was 0.03% (w / v) for the milk culture medium and 0.01% (w / v) for the whey culture medium.

[0112] Regarding BF-1, the number of viable cells in the culture medium heated simultaneously reached a maximum when the L-cystine concentrations in the skim milk culture medium and the whey culture medium were 0.1% (w / v) and 0.03% (w / v), respectively. In addition, the L-cysteine ​​concentration at which the number of viable cells in the culture medium heated simultaneously reached a maximum was 0.05% (w / v) in the skim milk culture medium and 0.03% (w / v) in the whey culture medium.

[0113] Regarding the concentration at which the viable count reaches the maximum, when n=3, separate heating and simultaneous heating are compared. The results are as follows: Figure 7As shown in Figures 8(BbY) and 8(BF-1), regardless of the strain and the additive, the viable cell count and acidity increased by simultaneous heating compared with heating separately. In particular, when BbY was cultured in a skim milk medium or whey medium supplemented with L-cystine or L-cysteine, and when BF-1 was cultured in a skim milk medium supplemented with L-cystine or whey medium supplemented with L-cystine or L-cysteine, it was confirmed that the viable cell count increased significantly by 1.3 to 2.6 times compared with heating separately.

[0114] The above results indicate that heating causes milk to react with L-cystine or L-cysteine ​​to produce a certain growth promoting factor.

[0115] Example 3 Changes in proliferation promoting effect due to whey separated from skim milk before and after heating and timing of cysteine ​​addition

[0116] (1) Test strains

[0117] Use BbY.

[0118] (2) Culture medium preparation

[0119] (i) Separation of whey culture medium before heating

[0120] The culture medium in which whey and L-cysteine ​​were separated before heating was prepared in the same manner as in Example 2. In addition, a culture medium to which L-cysteine ​​was added after heat sterilization and immediately before inoculation (separately heated culture medium) was also prepared. The added concentration of L-cysteine ​​was 0.03 (w / v) % as the final concentration.

[0121] (ii) Separation of whey culture medium after heating

[0122] 12% (w / v) skim milk obtained by dissolving skim milk powder in RO water to 12% (w / v) was heat sterilized in an autoclave at 115°C for 30 minutes under anaerobic conditions based on nitrogen, adjusted to pH 4.6 with 5N HCl, and centrifuged at 3000×g for 5 minutes. Then, the pH of the supernatant was adjusted to 6.5 with 5N NaOH, and centrifuged at 10000×g for 15 to 30 minutes. While replacing the gas phase in a pre-sterilized medium-sized test tube with a rim with nitrogen, the supernatant was dispensed in 10 mL portions, sealed with a butyl rubber stopper, and heated to prepare a separated whey medium. In addition, under anaerobic conditions, a medium in which L-cysteine ​​was added before heating and then heat sterilized (simultaneous heating medium) and a medium in which L-cysteine ​​was added after heat sterilization and before pH was adjusted to 4.6 (separate heating medium) were prepared in the same manner as the skim milk medium in Example 1.

[0123] (3) Inoculation and culture conditions

[0124] Inoculation and cultivation were carried out in the same manner as in Example 1.

[0125] (4) Various parameter measurement methods (i) pH, (ii) acidity, and (iii) viable cell count were measured in the same manner as in Example 1.

[0126] (iv) Ratio of viable bacteria

[0127] Based on the viable cell count of (iii), the viable cell count ratio was calculated by dividing the viable cell count in the simultaneously heated culture medium by the viable cell count in the separately heated culture medium.

[0128] (5) Results

[0129] The results are shown in Table 2 below.

[0130] [Table 2]

[0131]

[0132] Whether the casein was removed before heating (whey culture medium separated before heating) or the casein was removed after heating in a state containing casein (whey culture medium separated after heating), the proliferation of BbY was confirmed to be promoted by the addition of L-cysteine. In addition, in all cases, the number of viable bacteria in the culture medium heated simultaneously was higher than that in the culture medium heated separately, but the number of viable bacteria in the culture medium heated simultaneously and the culture medium heated separately was higher than that in the culture medium separated before heating, so it is considered that whey is more closely related to the proliferation promoting factor than casein.

[0133] Example 4 Changes in proliferation promoting effect caused by simultaneous heating temperature of milk and L-cystine

[0134] (1) Test strains

[0135] Use BbY.

[0136] (2) Culture medium preparation

[0137] (i) Skim milk culture medium

[0138] Except for the heating temperature and the added concentration of L-cystine, a skim milk medium and a L-cystine simultaneous heating medium and a separate heating medium were prepared in the same manner as in Example 1. Heating was performed at 115° C. for 30 minutes or at 121° C. for 30 minutes. Regarding the added concentration of L-cystine, L-cystine was added before or after heating so as to have a final concentration of 0.03% (w / v).

[0139] (3) Inoculation and culture conditions

[0140] Inoculation and cultivation were carried out in the same manner as in Example 1.

[0141] (4) Various parameter measurement methods

[0142] (i) pH, (ii) acidity, and (iii) viable cell count were measured in the same manner as in Example 1.

[0143] (5) Results

[0144] The results are shown in Table 3 below.

[0145] [Table 3]

[0146]

[0147] The promotion of BbY proliferation by the addition of L-cystine was confirmed at all heating temperatures. In addition, at all heating temperatures, it was confirmed that the number of viable cells and the acidity increased when skim milk and L-cystine were heated simultaneously compared to when skim milk and L-cystine were heated separately.

[0148] Example 5 Exploration of proliferation promoting factors

[0149] The results of Examples 1 to 4 indicate that heating causes milk to react with L-cystine or L-cysteine ​​to produce a growth-promoting factor. In addition, it is believed that the production of this growth-promoting factor is related to the components contained in the whey in the milk. Therefore, a search for this growth-promoting factor was conducted.

[0150] (1) Methods

[0151] L-cysteine ​​hydrochloride was added to the whey medium separated before heating at a final concentration of 0.03% (w / v), and heated at 115°C for 30 minutes. The pH of the obtained heat-treated product was adjusted to 8.0 by 5N NaOH, and centrifuged at 13000×g for 30 minutes. The supernatant was added to an ultrafiltration filter AmiconUltra-15 3kDa (manufactured by Merck) and centrifuged at 5000×g for 60 minutes. The filtrate (molecular weight fraction less than 3000) was subjected to liquid chromatography using an anion exchange column under the following conditions (the mobile phase was Tris buffer).

[0152] Column: HiTrap Q FF 1mL

[0153] Starting buffer: 20 mM Tris buffer (pH 8.0)

[0154] Dissolution buffer: 20 mM Tris buffer (pH 8.0) containing 1 M NaCl

[0155] Dissolution method: Flowthrough 20CV, Gradient (0→100%, 20CV), 2mL / fraction, wash (100%, 5CV)

[0156] Adding method: directly add 10mL of sample

[0157] Flow rate: 1mL / min

[0158] Column temperature: room temperature

[0159] Equipment: AKTA detector (GE HEALTHCARE)

[0160] 1 mL of the obtained fraction was added to Example 1 and 9 mL of the same skim milk medium after heating at 115°C for 30 minutes, and BbY was inoculated (the sample was heated at the same time). The viable cell count and acidity after culturing at 37°C for 12 hours were measured in the same manner as Example 1.

[0161] The same operation was also performed on a sample prepared by adding L-cysteine ​​hydrochloride to a pre-heated whey culture medium heated at 115°C for 30 minutes at a final concentration of 0.03% (w / v) (each sample was heated). As a control, 1 mL of Tris buffer was added to a skim milk culture medium to culture BbY.

[0162] (2) Results

[0163] exist Fig. 9 The viable count of the control sample (5.5×10 8 The ratio of the number of viable cells in each fraction when CFU / mL is set to 1 is Fig.10 The difference in acidity of each fraction when the acidity of the control sample is set to 0 is shown in FIG. In the samples heated simultaneously, the proliferation promoting effect was confirmed in the fraction with a dissolution volume of about 22 to 28 mL. On the other hand, no fraction with a proliferation promoting effect was confirmed in the samples heated separately. The fraction with a dissolution volume of 22 to 28 mL corresponds to the fraction eluted in the presence of 50 to 200 mM anions.

[0164] The same results were obtained even when BF-1 was used as the bacteria of the genus Bifidobacterium.

[0165] Therefore, it was clarified that the growth promoting factor of Bifidobacterium bacteria is a component with a molecular weight of less than 3000 in a heat-treated product of a milk culture medium containing cysteine, cystine or their salts, especially L-cysteine ​​hydrochloride, and is a milk culture medium component that is maintained on an anion exchange resin at a pH of 8.0 or above and eluted from the anion exchange resin in the presence of 50 to 200 mM anions.

Claims

1. A growth promoter for Bifidobacterium bacteria, comprising as an active ingredient a heat-treated product of a milk culture medium to which one or more selected from L-cysteine, L-cystine and salts thereof are added, The heat-treated product is a component of the heat-treated milk culture medium having a molecular weight of less than 3000, which is retained on an anion exchange resin at a pH of 8.0 or higher and eluted from the anion exchange resin in the presence of 50 to 200 mM anions. The milk culture medium is a skim milk culture medium or a whey culture medium.

2. The growth promoter according to claim 1, wherein The heating treatment is performed at 60 to 125° C. for 10 to 40 minutes.

3. The growth promoter according to claim 1 or 2, wherein The milk medium was supplemented with L-cysteine ​​hydrochloride.

4. A milk culture medium component having a molecular weight of less than 3000, which is a milk culture medium to which one or more selected from L-cysteine, L-cystine and salts thereof are added and which has been heated, the milk culture medium being retained on an anion exchange resin at a pH of 8.0 or higher and eluted from the anion exchange resin in the presence of 50 to 200 mM anions, wherein the milk culture medium is a skim milk culture medium or a whey culture medium.

5. A method for producing a milk culture medium component, characterized in that: A milk culture medium to which one or more selected from L-cysteine, L-cystine and their salts are added is heat-treated, and components having a molecular weight of less than 3000 are separated from the obtained heat-treated milk culture medium, and the obtained components are retained on an anion resin at a pH of 8.0 or above, and further eluted from the anion resin in the presence of 50 to 200 mM anions, wherein the milk culture medium is a skim milk culture medium or a whey culture medium.

Images